U.S. patent application number 09/866562 was filed with the patent office on 2002-01-24 for compositions and methods for the therapy and diagnosis of lung cancer.
Invention is credited to Bangur, Chaitanya S., Harlocker, Susan L., Klee, Jennifer I., Switzer, Ann, Wang, Tongtong.
Application Number | 20020009758 09/866562 |
Document ID | / |
Family ID | 26902278 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009758 |
Kind Code |
A1 |
Harlocker, Susan L. ; et
al. |
January 24, 2002 |
Compositions and methods for the therapy and diagnosis of lung
cancer
Abstract
Compositions and methods for the therapy and diagnosis of
cancer, particularly lung cancer, are disclosed. Illustrative
compositions comprise one or more lung tumor polypeptides,
immunogenic portions thereof, polynucleotides that encode such
polypeptides, antigen presenting cell that expresses such
polypeptides, and T cells that are specific for cells expressing
such polypeptides. The disclosed compositions are useful, for
example, in the diagnosis, prevention and/or treatment of diseases,
particularly lung cancer.
Inventors: |
Harlocker, Susan L.;
(Seattle, WA) ; Wang, Tongtong; (Medina, WA)
; Bangur, Chaitanya S.; (Seattle, WA) ; Klee,
Jennifer I.; (Seattle, WA) ; Switzer, Ann;
(Seattle, WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Family ID: |
26902278 |
Appl. No.: |
09/866562 |
Filed: |
May 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60207485 |
May 26, 2000 |
|
|
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60230475 |
Sep 6, 2000 |
|
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|
Current U.S.
Class: |
435/7.23 ;
424/93.7; 435/372.3; 435/69.3; 530/388.1; 536/23.5 |
Current CPC
Class: |
A61K 39/0011 20130101;
A61K 2039/505 20130101; C07K 2319/00 20130101; C12Q 1/6886
20130101; C07K 16/3023 20130101; A61P 35/00 20180101; C07K 14/47
20130101; A61K 2039/515 20130101 |
Class at
Publication: |
435/7.23 ;
435/69.3; 530/388.1; 536/23.5; 435/372.3; 424/93.7 |
International
Class: |
G01N 033/574; C07H
021/04; C12P 021/02; C12N 005/08; C07K 016/30 |
Claims
What is claimed:
1. An isolated polynucleotide comprising a sequence selected from
the group consisting of: (a) sequences provided in SEQ ID NO:1-3,
5, 7, 9, 11-19, 25-35, 44, 46, 47, 48, 53-55, 58-60, 66, 74, 75,
79, 81, 84, 85, 87, 93, 94 and 95; (b) complements of the sequences
provided in SEQ ID NO: 1-3, 5, 7, 9, 11-19, 25-35, 44, 46, 47, 48,
53-55, 58-60, 66, 74, 75, 79, 81, 84, 85, 87, 93, 94 and 95; (c)
sequences consisting of at least 20 contiguous residues of a
sequence provided in SEQ ID NO:1-3, 5, 7, 9, 11-19, 25-35, 44, 46,
47, 48, 53-55, 58-60, 66, 74, 75, 79, 81, 84, 85, 87, 93, 94 and
95; (d) sequences that hybridize to a sequence provided in SEQ ID
NO:1-3, 5, 7, 9, 11-19, 25-35, 44, 46, 47, 48, 53-55, 58-60, 66,
74, 75, 79, 81, 84, 85, 87, 93, 94 and 95, under highly stringent
conditions; (e) sequences having at least 75% identity to a
sequence of SEQ ID NO:1-3, 5, 7, 9, 11-19, 25-35, 44, 46, 47, 48,
53-55, 58-60, 66, 74, 75, 79, 81, 84, 85, 87, 93, 94 and 95; (f)
sequences having at least 90% identity to a sequence of SEQ ID
NO:1-3, 5, 7, 9, 11-19, 25-35, 44, 46, 47, 48, 53-55, 58-60, 66,
74, 75, 79, 81, 84, 85, 87, 93, 94 and 95; and (g) degenerate
variants of a sequence provided in SEQ ID NO:1-3, 5, 7, 9, 11-19,
25-35, 44, 46, 47, 48, 53-55, 58-60, 66, 74, 75, 79, 81, 84, 85,
87, 93, 94 and 95.
2. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) sequences having an
amino acid sequence of any one of SEQ ID NO:61, 62 and 96; (b)
sequences encoded by a polynucleotide of claim 1; (c) sequences
having at least 70% identity to a sequence encoded by a
polynucleotide of claim 1; and (d) sequences having at least 90%
identity to a sequence encoded by a polynucleotide of claim 1.
3. An expression vector comprising a polynucleotide of claim 1
operably linked to an expression control sequence.
4. A host cell transformed or transfected with an expression vector
according to claim 3.
5. An isolated antibody, or antigen-binding fragment thereof, that
specifically binds to a polypeptide of claim 2.
6. A method for detecting the presence of a cancer in a patient,
comprising the steps of: (a) obtaining a biological sample from the
patient; (b) contacting the biological sample with a binding agent
that binds to a polypeptide of claim 2; (c) detecting in the sample
an amount of polypeptide that binds to the binding agent; and (d)
comparing the amount of polypeptide to a predetermined cut-off
value and therefrom determining the presence of a cancer in the
patient.
7. A fusion protein comprising at least one polypeptide according
to claim 2.
8. An oligonucleotide that hybridizes to a sequence recited in SEQ
ID NO:1-3, 5, 7, 9, 11-19, 25-35, 44, 46, 47, 48, 53-55, 58-60, 66,
74, 75, 79, 81, 84, 85, 87, 93, 94 and 95 under highly stringent
conditions.
9. A method for stimulating and/or expanding T cells specific for a
tumor protein, comprising contacting T cells with at least one
component selected from the group consisting of: (a) polypeptides
according to claim 2; (b) polynucleotides according to claim 1; and
(c) polynucleotides having a nucleotide sequence of any one of SEQ
ID NO:4, 6, 8, 10, 20-24, 42, 43, 45, 49-52, 63-65, 67-73, 76-78,
80, 82, 83, 86 and 88-91; (d) antigen-presenting cells that express
a polynucleotide according to claim 1, under conditions and for a
time sufficient to permit the stimulation and/or expansion of T
cells.
10. An isolated T cell population, comprising T cells prepared
according to the method of claim 9.
11. A composition comprising a first component selected from the
group consisting of physiologically acceptable carriers and
immunostimulants, and a second component selected from the group
consisting of: (a) polypeptides according to claim 2; (b)
polynucleotides according to claim 1; (c) polynucleotides having a
nucleotide sequence of any one of SEQ ID NO:4, 6, 8, 10, 20-24, 42,
43, 45, 49-52, 63-65, 67-73, 76-78, 80, 82, 83, 86 and 88-91; (d)
antibodies according to claim 5; (e) fusion proteins according to
claim 7; (f) T cell populations according to claim 10; and (g)
antigen presenting cells that express a polypeptide according to
claim 2.
12. A method for stimulating an immune response in a patient,
comprising administering to the patient a composition of claim
11.
13. A method for the treatment of a lung cancer in a patient,
comprising administering to the patient a composition of claim
11.
14. A method for determining the presence of a cancer in a patient,
comprising the steps of: (a) obtaining a biological sample from the
patient; (b) contacting the biological sample with an
oligonucleotide according to claim 8; (c) detecting in the sample
an amount of a polynucleotide that hybridizes to the
oligonucleotide; and (d) compare the amount of polynucleotide that
hybridizes to the oligonucleotide to a predetermined cut-off value,
and therefrom determining the presence of the cancer in the
patient.
15. A diagnostic kit comprising at least one oligonucleotide
according to claim 8.
16. A diagnostic kit comprising at least one antibody according to
claim 5 and a detection reagent, wherein the detection reagent
comprises a reporter group.
17. A method for the treatment of lung cancer in a patient,
comprising the steps of: (a) incubating CD4+ and/or CD8+ T cells
isolated from a patient with at least one component selected from
the group consisting of: (i) polypeptides according to claim 2;
(ii) polynucleotides according to claim 1; and (iii) antigen
presenting cells that express a polypeptide of claim 2, such that T
cell proliferate; (b) administering to the patient an effective
amount of the proliferated T cells, and thereby inhibiting the
development of a cancer in the patient.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to U.S. Provisional Application
No. 60/207,485, filed May 26, 2000 and U.S. Provisional Application
No. 60/230,475, filed Sep. 6, 2000, incorporated in their entirety
herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates generally to therapy and
diagnosis of cancer, such as lung cancer. The invention is more
specifically related to polypeptides, comprising at least a portion
of a lung tumor protein, and to polynucleotides encoding such
polypeptides. Such polypeptides and polynucleotides are useful in
pharmaceutical compositions, e.g., vaccines, and other compositions
for the diagnosis and treatment of lung cancer.
BACKGROUND OF THE INVENTION
[0003] Cancer is a significant health problem throughout the world.
Although advances have been made in detection and therapy of
cancer, no vaccine or other universally successful method for
prevention and/or treatment is currently available. Current
therapies, which are generally based on a combination of
chemotherapy or surgery and radiation, continue to prove inadequate
in many patients.
[0004] Lung cancer is a significant health problem throughout the
world. In the U.S., lung cancer is the primary cause of cancer
death among both men and women, with an estimated 172,000 new cases
being reported in 1994. The five-year survival rate among all lung
cancer patients, regardless of the stage of disease at diagnosis,
is only 13%. This contrasts with a five-year survival rate of 46%
among cases detected while the disease is still localized. However,
early detection of lung cancer is difficult since clinical symptoms
are often not seen until the disease has reached an advanced stage,
and only 16% of lung cancers are discovered before the disease has
spread.
[0005] In spite of considerable research into therapies for these
and other cancers, lung cancer remains difficult to diagnose and
treat effectively. Accordingly, there is a need in the art for
improved methods for detecting and treating such cancers. The
present invention fulfills these needs and further provides other
related advantages.
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention provides polynucleotide
compositions comprising a sequence selected from the group
consisting of:
[0007] (a) sequences provided in SEQ ID NO:1-35, 42-55, 58-60,
63-91 and 93-95;
[0008] (b) complements of the sequences provided in SEQ ID NO:1-35,
42-55, 58-60, 63-91 and 93-95;
[0009] (c) sequences consisting of at least 20, 25, 30, 35, 40, 45,
50, 75 and 100 contiguous residues of a sequence provided in SEQ ID
NO: 1-35, 42-55, 58-60, 63-91 and 93-95;
[0010] (d) sequences that hybridize to a sequence provided in SEQ
ID NO: 1-35, 42-55, 58-60, 63-91 and 93-95, under moderate or
highly stringent conditions;
[0011] (e) sequences having at least 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98% or 99% identity to a sequence of SEQ ID NO:1-35, 42-55,
58-60, 63-91 and 93-95; and
[0012] (f) degenerate variants of a sequence provided in SEQ ID NO:
1-35, 42-55, 58-60, 63-91 and 93-95.
[0013] In one preferred embodiment, the polynucleotide compositions
of the invention are expressed in at least about 20%, more
preferably in at least about 30%, and most preferably in at least
about 50% of lung tumors samples tested, at a level that is at
least about 2-fold, preferably at least about 5-fold, and most
preferably at least about 10-fold higher than that for normal
tissues.
[0014] The present invention, in another aspect, provides
polypeptide compositions comprising an amino acid sequence that is
encoded by a polynucleotide sequence described above.
[0015] The present invention further provides polypeptide
compositions comprising an amino acid sequence selected from the
group consisting of sequences recited in SEQ ID NO:36-41, 56, 57,
61, 62, 92 and 96.
[0016] In certain preferred embodiments, the polypeptides and/or
polynucleotides of the present invention are immunogenic, i.e.,
they are capable of eliciting an immune response, particularly a
humoral and/or cellular immune response, as further described
herein.
[0017] The present invention further provides fragments, variants
and/or derivatives of the disclosed polypeptide and/or
polynucleotide sequences, wherein the fragments, variants and/or
derivatives preferably have a level of immunogenic activity of at
least about 50%, preferably at least about 70% and more preferably
at least about 90% of the level of immunogenic activity of a
polypeptide sequence set forth in SEQ ID NO:36-41, 56, 57, 61, 62,
92 and 96 or a polypeptide sequence encoded by a polynucleotide
sequence set forth in SEQ ID NO:1-35, 42-55, 58-60, 63-91 and
93-95.
[0018] The present invention further provides polynucleotides that
encode a polypeptide described above, expression vectors comprising
such polynucleotides and host cells transformed or transfected with
such expression vectors.
[0019] Within other aspects, the present invention provides
pharmaceutical compositions comprising a polypeptide or
polynucleotide as described above and a physiologically acceptable
carrier.
[0020] Within a related aspect of the present invention, the
pharmaceutical compositions, e.g., vaccine compositions, are
provided for prophylactic or therapeutic applications. Such
compositions generally comprise an immunogenic polypeptide or
polynucleotide of the invention and an immunostimulant, such as an
adjuvant.
[0021] The present invention further provides pharmaceutical
compositions that comprise: (a) an antibody or antigen-binding
fragment thereof that specifically binds to a polypeptide of the
present invention, or a fragment thereof; and (b) a physiologically
acceptable carrier.
[0022] Within further aspects, the present invention provides
pharmaceutical compositions comprising: (a) an antigen presenting
cell that expresses a polypeptide as described above and (b) a
pharmaceutically acceptable carrier or excipient. Illustrative
antigen presenting cells include dendritic cells, macrophages,
monocytes, fibroblasts and B cells.
[0023] Within related aspects, pharmaceutical compositions are
provided that comprise: (a) an antigen presenting cell that
expresses a polypeptide as described above and (b) an
immunostimulant.
[0024] The present invention further provides, in other aspects,
fusion proteins that comprise at least one polypeptide as described
above, as well as polynucleotides encoding such fusion proteins,
typically in the form of pharmaceutical compositions, e.g., vaccine
compositions, comprising a physiologically acceptable carrier
and/or an immunostimulant. The fusions proteins may comprise
multiple immunogenic polypeptides or portions/variants thereof, as
described herein, and may further comprise one or more polypeptide
segments for facilitating the expression, purification and/or
immunogenicity of the polypeptide(s).
[0025] Within further aspects, the present invention provides
methods for stimulating an immune response in a patient, preferably
a T cell response in a human patient, comprising administering a
pharmaceutical composition described herein. The patient may be
afflicted with lung cancer, in which case the methods provide
treatment for the disease, or patient considered at risk for such a
disease may be treated prophylactically.
[0026] Within further aspects, the present invention provides
methods for inhibiting the development of a cancer in a patient,
comprising administering to a patient a pharmaceutical composition
as recited above. The patient may be afflicted with lung cancer, in
which case the methods provide treatment for the disease, or
patient considered at risk for such a disease may be treated
prophylactically.
[0027] The present invention further provides, within other
aspects, methods for removing tumor cells from a biological sample,
comprising contacting a biological sample with T cells that
specifically react with a polypeptide of the present invention,
wherein the step of contacting is performed under conditions and
for a time sufficient to permit the removal of cells expressing the
protein from the sample.
[0028] Within related aspects, methods are provided for inhibiting
the development of a cancer in a patient, comprising administering
to a patient a biological sample treated as described above.
[0029] Methods are further provided, within other aspects, for
stimulating and/or expanding T cells specific for a polypeptide of
the present invention, comprising contacting T cells with one or
more of: (i) a polypeptide as described above; (ii) a
polynucleotide encoding such a polypeptide; and/or (iii) an antigen
presenting cell that expresses such a polypeptide; under conditions
and for a time sufficient to permit the stimulation and/or
expansion of T cells. Isolated T cell populations comprising T
cells prepared as described above are also provided.
[0030] Within further aspects, the present invention provides
methods for inhibiting the development of a cancer in a patient,
comprising administering to a patient an effective amount of a T
cell population as described above.
[0031] The present invention further provides methods for
inhibiting the development of a cancer in a patient, comprising the
steps of: (a) incubating CD4.sup.+ and/or CD8.sup.+ T cells
isolated from a patient with one or more of: (i) a polypeptide
comprising at least an immunogenic portion of polypeptide disclosed
herein; (ii) a polynucleotide encoding such a polypeptide; and
(iii) an antigen-presenting cell that expressed such a polypeptide;
and (b) administering to the patient an effective amount of the
proliferated T cells, and thereby inhibiting the development of a
cancer in the patient. Proliferated cells may, but need not, be
cloned prior to administration to the patient.
[0032] Within further aspects, the present invention provides
methods for determining the presence or absence of a cancer,
preferably a lung cancer, in a patient comprising: (a) contacting a
biological sample obtained from a patient with a binding agent that
binds to a polypeptide as recited above; (b) detecting in the
sample an amount of polypeptide that binds to the binding agent;
and (c) comparing the amount of polypeptide with a predetermined
cut-off value, and therefrom determining the presence or absence of
a cancer in the patient. Within preferred embodiments, the binding
agent is an antibody, more preferably a monoclonal antibody.
[0033] The present invention also provides, within other aspects,
methods for monitoring the progression of a cancer in a patient.
Such methods comprise the steps of: (a) contacting a biological
sample obtained from a patient at a first point in time with a
binding agent that binds to a polypeptide as recited above; (b)
detecting in the sample an amount of polypeptide that binds to the
binding agent; (c) repeating steps (a) and (b) using a biological
sample obtained from the patient at a subsequent point in time; and
(d) comparing the amount of polypeptide detected in step (c) with
the amount detected in step (b) and therefrom monitoring the
progression of the cancer in the patient.
[0034] The present invention further provides, within other
aspects, methods for determining the presence or absence of a
cancer in a patient, comprising the steps of: (a) contacting a
biological sample, e.g., tumor sample, serum sample, etc., obtained
from a patient with an oligonucleotide that hybridizes to a
polynucleotide that encodes a polypeptide of the present invention;
(b) detecting in the sample a level of a polynucleotide, preferably
mRNA, that hybridizes to the oligonucleotide; and (c) comparing the
level of polynucleotide that hybridizes to the oligonucleotide with
a predetermined cut-off value, and therefrom determining the
presence or absence of a cancer in the patient. Within certain
embodiments, the amount of mRNA is detected via polymerase chain
reaction using, for example, at least one oligonucleotide primer
that hybridizes to a polynucleotide encoding a polypeptide as
recited above, or a complement of such a polynucleotide. Within
other embodiments, the amount of mRNA is detected using a
hybridization technique, employing an oligonucleotide probe that
hybridizes to a polynucleotide that encodes a polypeptide as
recited above, or a complement of such a polynucleotide.
[0035] In related aspects, methods are provided for monitoring the
progression of a cancer in a patient, comprising the steps of: (a)
contacting a biological sample obtained from a patient with an
oligonucleotide that hybridizes to a polynucleotide that encodes a
polypeptide of the present invention; (b) detecting in the sample
an amount of a polynucleotide that hybridizes to the
oligonucleotide; (c) repeating steps (a) and (b) using a biological
sample obtained from the patient at a subsequent point in time; and
(d) comparing the amount of polynucleotide detected in step (c)
with the amount detected in step (b) and therefrom monitoring the
progression of the cancer in the patient.
[0036] Within further aspects, the present invention provides
antibodies, such as monoclonal antibodies, that bind to a
polypeptide as described above, as well as diagnostic kits
comprising such antibodies. Diagnostic kits comprising one or more
oligonucleotide probes or primers as described above are also
provided.
[0037] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entirety as if each was
incorporated individually.
SEQUENCE IDENTIFIERS
[0038] SEQ ID NO: 1 is the cDNA sequence for Clone ID # 55964 which
is named clone L1040C, and is the same sequence as SEQ ID NO:2337
from U.S. Provisional Application No. 60/207,485.
[0039] SEQ ID NO:2 is an extended cDNA sequence for L1040C (Clone
ID # 55964).
[0040] SEQ ID NO:3 is the cDNA sequence for Clone ID # 58269 which
is named clone L1039C, and is the same sequence as SEQ ID NO:7264
from U.S. Provisional Application No. 60/207,485.
[0041] SEQ ID NO:4 is an extended cDNA sequence for L1039C (Clone
ID # 58269), and which corresponds to the fbx5 F-box gene.
[0042] SEQ ID NO:5 is the cDNA sequence for Clone ID # 58267 which
is named clone L1037C, and is the same sequence as SEQ ID NO:4978
from U.S. Provisional Application No. 60/207,485.
[0043] SEQ ID NO:6 is an extended cDNA sequence for L1037C (Clone #
58267), and which corresponds to the mitotic checkpoint kinase
mad3-like gene.
[0044] SEQ ID NO:7 is the cDNA sequence for Clone ID # 58245 which
is named clone L1038C, and is the same sequence as SEQ ID NO:1796
from U.S. Provisional Application No. 60/207,485.
[0045] SEQ ID NO:8 is an extended cDNA sequence for L1038C (Clone
ID # 58245), and which corresponds to a neuronal ER localized
gene.
[0046] SEQ ID NO:9 is the cDNA sequence for Clone ID # 55571 which
is named clone L1027C, and is the same sequence as SEQ ID NO:4538
from U.S. Provisional Application No. 60/207,485.
[0047] SEQ ID NO:10 is an extended cDNA sequence for L1027C (Clone
ID # 55571).
[0048] SEQ ID NO: 11 is the cDNA sequence for Clone ID # 55978.
[0049] SEQ ID NO:12 is an extended cDNA sequence for Clone ID #
55978.
[0050] SEQ ID NO:13 is the cDNA sequence for Clone ID # 55980.
[0051] SEQ ID NO:14 is an extended cDNA sequence for Clone ID #
55980.
[0052] SEQ ID NO:15 is the cDNA sequence for Clone ID # 58346.
[0053] SEQ ID NO:16 is an extended cDNA sequence for Clone ID #
58346.
[0054] SEQ ID NO:17 is the cDNA sequence for Clone ID # 55561.
[0055] SEQ ID NO: 18 is an extended cDNA sequence for Clone ID #
55561.
[0056] SEQ ID NO:19 is the cDNA sequence for Clone ID # 55984.
[0057] SEQ ID NO:20 is an extended cDNA sequence for Clone ID #
55984, and which corresponds to a gt mismatch glycosylase gene.
[0058] SEQ ID NO:21 is the cDNA sequence for Clone ID # 58261.
[0059] SEQ ID NO:22 is an extended cDNA sequence for Clone ID #
58261, and which corresponds to a phosphoserine aminotransferase
gene.
[0060] SEQ ID NO:23 is the cDNA sequence for Clone ID # 58348.
[0061] SEQ ID NO:24 is an extended cDNA sequence for Clone ID #
58348, and which corresponds to a hCAP gene.
[0062] SEQ ID NO:25 is the cDNA sequence for Clone ID # 56016.
[0063] SEQ ID NO:26 is an extended cDNA sequence for Clone ID #
56016.
[0064] SEQ ID NO:27 is the cDNA sequence for Clone ID # 55987.
[0065] SEQ ID NO:28 is an extended cDNA sequence for Clone ID #
55987.
[0066] SEQ ID NO:29 is the cDNA sequence for Clone ID # 55956.
[0067] SEQ ID NO:30 is an extended cDNA sequence for Clone ID #
55956.
[0068] SEQ ID NO:31 is the cDNA sequence for Clone ID # 55952.
[0069] SEQ ID NO:32 is the cDNA sequence for Clone ID # 55957.
[0070] SEQ ID NO:33 is an extended cDNA sequence for Clone ID #
55957.
[0071] SEQ ID NO:34 is the cDNA sequence for Clone ID # 55559.
[0072] SEQ ID NO:35 is an extended cDNA sequence for Clone ID #
55559.
[0073] SEQ ID NO:36 is an amino acid sequence of an ORF for L1027C,
encoded by the polynucleotide of SEQ ID NO: 10.
[0074] SEQ ID NO:37 is an amino acid sequence of the F-box protein
Fbx5 encoded by SEQ ID NO:4.
[0075] SEQ ID NO:38 is an amino acid sequence of the mitotic
checkpoint kinase MAD3-like protein encoded by SEQ ID NO:6.
[0076] SEQ ID NO:39 is an amino acid sequence of the neuronal
olfactomedin-related ER localized protein encoded by SEQ ID
NO:8.
[0077] SEQ ID NO:40 is an amino acid sequence of the phosphoserine
aminotransferase encoded by SEQ ID NO:22.
[0078] SEQ ID NO:41 is an amino acid sequence of the gt mismatch
glycosylase encoded by SEQ ID NO:20.
[0079] SEQ ID NO:42 is the determined cDNA sequence for Clone ID #
63575 which is named clone L1053 C.
[0080] SEQ ID NO:43 is the determined cDNA sequence for Clone ID #
63582 which is named clone L1054C.
[0081] SEQ ID NO:44 is the determined cDNA sequence for Clone ID #
63598 which is named clone L1055C.
[0082] SEQ ID NO:45 is the determined cDNA sequence for Clone ID #
64963 which is named clone L1056C.
[0083] SEQ ID NO:46 is the determined cDNA sequence for Clone ID #
64988 which is named clone L1058C.
[0084] SEQ ID NO:47 is the determined cDNA sequence for Clone ID #
63485.
[0085] SEQ ID NO:48 is the determined cDNA sequence for Clone ID #
65010.
[0086] SEQ ID NO:49 is a predicted full-length cDNA sequence for
SEQ ID NO:42 which is a full-length sequence from Genbank for an
insulinoma-associated 1 mRNA.
[0087] SEQ ID NO:50 is a predicted full-length cDNA sequence for
SEQ ID NO:43 which is a full-length sequence from Genbank for
KIAA0535.
[0088] SEQ ID NO:51 is a predicted extended cDNA sequence for SEQ
ID NO:44.
[0089] SEQ ID NO:52 is a a predicted full-length cDNA sequence for
SEQ ID NO:45 which is a full-length sequence from genbank for a
human DAZ mRNA 3'UTR.
[0090] SEQ ID NO:53 is a predicted extended cDNA sequence for SEQ
ID NO:46.
[0091] SEQ ID NO:54 is a predicted extended cDNA sequence for SEQ
ID NO:47.
[0092] SEQ ID NO:55 is a predicted extended cDNA sequence for SEQ
ID NO:48.
[0093] SEQ ID NO:56 is the deduced amino acid sequence encoded by
SEQ ID NO:49.
[0094] SEQ ID NO:57 is the deduced amino acid sequence encoded by
SEQ ID NO:50.
[0095] SEQ ID NO:58 is the determined full-length cDNA sequence for
clone L1058C (sequence of the originally isolated clone is given in
SEQ ID NO:46 and the predicted extended cDNA sequence in SEQ ID
NO:53).
[0096] SEQ ID NO:59 is a first predicted ORF of SEQ ID NO:58.
[0097] SEQ ID NO:60 is a second predicted ORF of SEQ ID NO:58.
[0098] SEQ ID NO:61 is the deduced amino acid sequence encoded by
SEQ ID NO:59.
[0099] SEQ ID NO:62 is the deduced amino acid sequence encoded by
SEQ ID NO:60.
[0100] SEQ ID NO:63 is the determined cDNA sequence for Clone ID #
72761.
[0101] SEQ ID NO:64 is the determined cDNA sequence for Clone ID #
72762.
[0102] SEQ ID NO:65 is the determined cDNA sequence for Clone ID #
72763.
[0103] SEQ ID NO:66 is the determined cDNA sequence for Clone ID #
72764.
[0104] SEQ ID NO:67 is the determined cDNA sequence for Clone ID #
72765.
[0105] SEQ ID NO:68 is the determined cDNA sequence for Clone ID #
72766.
[0106] SEQ ID NO:69 is the determined cDNA sequence for Clone ID #
72772.
[0107] SEQ ID NO:70 is the determined cDNA sequence for Clone ID #
72775.
[0108] SEQ ID NO:71 is the determined cDNA sequence for Clone ID #
72776.
[0109] SEQ ID NO:72 is the determined cDNA sequence for Clone ID #
72779.
[0110] SEQ ID NO:73 is the determined cDNA sequence for Clone ID #
72781.
[0111] SEQ ID NO:74 is the determined cDNA sequence for Clone ID #
72784.
[0112] SEQ ID NO:75 is the determined cDNA sequence for Clone ID #
72788.
[0113] SEQ ID NO:76 is the determined cDNA sequence for Clone ID #
72789.
[0114] SEQ ID NO:77 is the determined cDNA sequence for Clone ID
72790.
[0115] SEQ ID NO:78 is the determined cDNA sequence for Clone ID #
72791.
[0116] SEQ ID NO:79 is the determined cDNA sequence for Clone ID #
72792.
[0117] SEQ ID NO:80 is the determined cDNA sequence for Clone ID
72794.
[0118] SEQ ID NO:81 is the determined cDNA sequence for Clone ID #
72795.
[0119] SEQ ID NO: 82 is the determined cDNA sequence for Clone ID
#72797.
[0120] SEQ ID NO:83 is the determined cDNA sequence for Clone ID #
72798.
[0121] SEQ ID NO:84 is the determined cDNA sequence for Clone ID #
72804.
[0122] SEQ ID NO:85 is the determined cDNA sequence for Clone ID #
72805.
[0123] SEQ ID NO:86 is the determined cDNA sequence for Clone ID #
72806.
[0124] SEQ ID NO:87 is the determined cDNA sequence for Clone ID #
72807.
[0125] SEQ ID NO:88 is the determined CDNA sequence for Clone ID #
72808.
[0126] SEQ ID NO:89 is the determined cDNA sequence for Clone ID #
72809.
[0127] SEQ ID NO:90 is the determined cDNA sequence for Clone ID #
72811.
[0128] SEQ ID NO:91 is the determined full-length cDNA sequence for
Clone ID 72813 which is named clone L1080C.
[0129] SEQ ID NO:92 is the deduced amino acid sequence encoded by
SEQ ID NO:91.
[0130] SEQ ID NO:93 is the ORF for L1027C.
[0131] SEQ ID NO:94 is a first determined full-length cDNA sequence
for L1027C.
[0132] SEQ ID NO:95 is a second determined full-length cDNA
sequence for L1027C.
[0133] SEQ ID NO:96 is the deduced amino acid sequence encoded by
SEQ ID NO:93.
DETAILED DESCRIPTION OF THE INVENTION
[0134] The present invention is directed generally to compositions
and their use in the therapy and diagnosis of cancer, particularly
lung cancer. As described further below, illustrative compositions
of the present invention include, but are not restricted to,
polypeptides, particularly immunogenic polypeptides,
polynucleotides encoding such polypeptides, antibodies and other
binding agents, antigen presenting cells (APCs) and immune system
cells (e.g., T cells).
[0135] The practice of the present invention will employ, unless
indicated specifically to the contrary, conventional methods of
virology, immunology, microbiology, molecular biology and
recombinant DNA techniques within the skill of the art, many of
which are described below for the purpose of illustration. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory
Manual (1982); DNA Cloning: A Practical Approach, vol. I & II
(D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984);
Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);
Transcription and Translation (B. Hames & S. Higgins, eds.,
1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A
Practical Guide to Molecular Cloning (1984).
[0136] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0137] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
[0138] Polypeptide Compositions
[0139] As used herein, the term "polypeptide" "is used in its
conventional meaning, i.e., as a sequence of amino acids. The
polypeptides are not limited to a specific length of the product;
thus, peptides, oligopeptides, and proteins are included within the
definition of polypeptide, and such terms may be used
interchangeably herein unless specifically indicated otherwise.
This term also does not refer to or exclude post-expression
modifications of the polypeptide, for example, glycosylations,
acetylations, phosphorylations and the like, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring. A polypeptide may be an entire protein, or
a subsequence thereof. Particular polypeptides of interest in the
context of this invention are amino acid subsequences comprising
epitopes, i.e., antigenic determinants substantially responsible
for the immunogenic properties of a polypeptide and being capable
of evoking an immune response.
[0140] Particularly illustrative polypeptides of the present
invention comprise those encoded by a polynucleotide sequence set
forth in any one of SEQ ID NO:1-35, 42-55, 58-60, 63-91 and 93-95,
or a sequence that hybridizes under moderately stringent
conditions, or, alternatively, under highly stringent conditions,
to a polynucleotide sequence set forth in any one of SEQ ID
NO:1-35, 42-55, 58-60, 63-91 and 93-95. Certain other illustrative
polypeptides of the invention comprise amino acid sequences as set
forth in any one of SEQ ID NOs:36-41, 56, 57, 61, 62, 92 and
96.
[0141] The polypeptides of the present invention are sometimes
herein referred to as lung tumor proteins or lung tumor
polypeptides, as an indication that their identification has been
based at least in part upon their increased levels of expression in
lung tumor samples. Thus, a "lung tumor polypeptide" or "lung tumor
protein," refers generally to a polypeptide sequence of the present
invention, or a polynucleotide sequence encoding such a
polypeptide, that is expressed in a substantial proportion of lung
tumor samples, for example preferably greater than about 20%, more
preferably greater than about 30%, and most preferably greater than
about 50% or more of lung tumor samples tested, at a level that is
at least two fold, and preferably at least five fold, greater than
the level of expression in normal tissues, as determined using a
representative assay provided herein. A lung tumor polypeptide
sequence of the invention, based upon its increased level of
expression in tumor cells, has particular utility both as a
diagnostic marker as well as a therapeutic target, as further
described below.
[0142] In certain preferred embodiments, the polypeptides of the
invention are immunogenic, i.e., they react detectably within an
immunoassay (such as an ELISA or T-cell stimulation assay) with
antisera and/or T-cells from a patient with lung cancer. Screening
for immunogenic activity can be performed using techniques well
known to the skilled artisan. For example, such screens can be
performed using methods such as those described in Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988. In one illustrative example, a polypeptide may be immobilized
on a solid support and contacted with patient sera to allow binding
of antibodies within the sera to the immobilized polypeptide.
Unbound sera may then be removed and bound antibodies detected
using, for example, .sup.125I-labeled Protein A.
[0143] As would be recognized by the skilled artisan, immunogenic
portions of the polypeptides disclosed herein are also encompassed
by the present invention. An "immunogenic portion," as used herein,
is a fragment of an immunogenic polypeptide of the invention that
itself is immunologically reactive (i.e., specifically binds) with
the B-cells and/or T-cell surface antigen receptors that recognize
the polypeptide. Immunogenic portions may generally be identified
using well known techniques, such as those summarized in Paul,
Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and
references cited therein. Such techniques include screening
polypeptides for the ability to react with antigen-specific
antibodies, antisera and/or T-cell lines or clones. As used herein,
antisera and antibodies are "antigen-specific" if they specifically
bind to an antigen (i.e., they react with the protein in an ELISA
or other immunoassay, and do not react detectably with unrelated
proteins). Such antisera and antibodies may be prepared as
described herein, and using well-known techniques.
[0144] In one preferred embodiment, an immunogenic portion of a
polypeptide of the present invention is a portion that reacts with
antisera and/or T-cells at a level that is not substantially less
than the reactivity of the full-length polypeptide (e.g., in an
ELISA and/or T-cell reactivity assay). Preferably, the level of
immunogenic activity of the immunogenic portion is at least about
50%, preferably at least about 70% and most preferably greater than
about 90% of the immunogenicity for the full-length polypeptide. In
some instances, preferred immunogenic portions will be identified
that have a level of immunogenic activity greater than that of the
corresponding full-length polypeptide, e.g., having greater than
about 100% or 150% or more immunogenic activity.
[0145] In certain other embodiments, illustrative immunogenic
portions may include peptides in which an N-terminal leader
sequence and/or transmembrane domain have been deleted. Other
illustrative immunogenic portions will contain a small N- and/or
C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino
acids), relative to the mature protein.
[0146] In another embodiment, a polypeptide composition of the
invention may also comprise one or more polypeptides that are
immunologically reactive with T cells and/or antibodies generated
against a polypeptide of the invention, particularly a polypeptide
having an amino acid sequence disclosed herein, or to an
immunogenic fragment or variant thereof.
[0147] In another embodiment of the invention, polypeptides are
provided that comprise one or more polypeptides that are capable of
eliciting T cells and/or antibodies that are immunologically
reactive with one or more polypeptides described herein, or one or
more polypeptides encoded by contiguous nucleic acid sequences
contained in the polynucleotide sequences disclosed herein, or
immunogenic fragments or variants thereof, or to one or more
nucleic acid sequences which hybridize to one or more of these
sequences under conditions of moderate to high stringency.
[0148] The present invention, in another aspect, provides
polypeptide fragments comprising at least about 5, 10, 15, 20, 25,
50, or 100 contiguous amino acids, or more, including all
intermediate lengths, of a polypeptide compositions set forth
herein, such as those set forth in SEQ ID NOs:36-41, 56, 57, 61,
62, 92 and 96, or those encoded by a polynucleotide sequence set
forth in a sequence of SEQ ID NOs:1-35, 42-55, 58-60, 63-91 and
93-95.
[0149] In another aspect, the present invention provides variants
of the polypeptide compositions described herein. Polypeptide
variants generally encompassed by the present invention will
typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined
as described below), along its length, to a polypeptide sequences
set forth herein.
[0150] In one preferred embodiment, the polypeptide fragments and
variants provided by the present invention are immunologically
reactive with an antibody and/or T-cell that react with a
full-length polypeptide specifically set forth herein.
[0151] In another preferred embodiment, the polypeptide fragments
and variants provided by the present invention exhibit a level of
immunogenic activity of at least about 50%, preferably at least
about 70%, and most preferably at least about 90% or more of that
exhibited by a full-length polypeptide sequence specifically set
forth herein.
[0152] A polypeptide "variant," as the term is used herein, is a
polypeptide that typically differs from a polypeptide specifically
disclosed herein in one or more substitutions, deletions, additions
and/or insertions. Such variants may be naturally occurring or may
be synthetically generated, for example, by modifying one or more
of the above polypeptide sequences of the invention and evaluating
their immunogenic activity as described herein and/or using any of
a number of techniques well known in the art.
[0153] For example, certain illustrative variants of the
polypeptides of the invention include those in which one or more
portions, such as an N-terminal leader sequence or transmembrane
domain, have been removed. Other illustrative variants include
variants in which a small portion (e.g., 1-30 amino acids,
preferably 5-15 amino acids) has been removed from the N- and/or
C-terminal of the mature protein.
[0154] In many instances, a variant will contain conservative
substitutions. A "conservative substitution" is one in which an
amino acid is substituted for another amino acid that has similar
properties, such that one skilled in the art of peptide chemistry
would expect the secondary structure and hydropathic nature of the
polypeptide to be substantially unchanged. As described above,
modifications may be made in the structure of the polynucleotides
and polypeptides of the present invention and still obtain a
functional molecule that encodes a variant or derivative
polypeptide with desirable characteristics, e.g., with immunogenic
characteristics. When it is desired to alter the amino acid
sequence of a polypeptide to create an equivalent, or even an
improved, immunogenic variant or portion of a polypeptide of the
invention, one skilled in the art will typically change one or more
of the codons of the encoding DNA sequence according to Table
1.
[0155] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like
properties. It is thus contemplated that various changes may be
made in the peptide sequences of the disclosed compositions, or
corresponding DNA sequences which encode said peptides without
appreciable loss of their biological utility or activity.
1TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K
AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0156] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporated herein by reference). It is accepted that the relative
hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like. Each amino acid has been assigned a hydropathic index on the
basis of its hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982). These values are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0157] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e. still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred. It is also understood in the
art that the substitution of like amino acids can be made
effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101
(specifically incorporated herein by reference in its entirety),
states that the greatest local average hydrophilicity of a protein,
as governed by the hydrophilicity of its adjacent amino acids,
correlates with a biological property of the protein.
[0158] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent protein. In such
changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0159] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
that take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0160] In addition, any polynucleotide may be further modified to
increase stability in vivo. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends; the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages in the backbone; and/or the inclusion of
nontraditional bases such as inosine, queosine and wybutosine, as
well as acetyl- methyl-, thio- and other modified forms of adenine,
cytidine, guanine, thymine and uridine.
[0161] Amino acid substitutions may further be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and
valine; glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain nonconservative
changes. In a preferred embodiment, variant polypeptides differ
from a native sequence by substitution, deletion or addition of
five amino acids or fewer. Variants may also (or alternatively) be
modified by, for example, the deletion or addition of amino acids
that have minimal influence on the immunogenicity, secondary
structure and hydropathic nature of the polypeptide.
[0162] As noted above, polypeptides may comprise a signal (or
leader) sequence at the N-terminal end of the protein, which
co-translationally or post-translationally directs transfer of the
protein. The polypeptide may also be conjugated to a linker or
other sequence for ease of synthesis, purification or
identification of the polypeptide (e.g., poly-His), or to enhance
binding of the polypeptide to a solid support. For example, a
polypeptide may be conjugated to an immunoglobulin Fc region.
[0163] When comparing polypeptide sequences, two sequences are said
to be "identical" if the sequence of amino acids in the two
sequences is the same when aligned for maximum correspondence, as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, 40 to about
50, in which a sequence may be compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned.
[0164] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E.
W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971)
Comb. Theor 11:105; Saitou, N. Nei, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
[0165] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0166] One preferred example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0
can be used, for example with the parameters described herein, to
determine percent sequence identity for the polynucleotides and
polypeptides of the invention. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. For amino acid sequences, a scoring
matrix can be used to calculate the cumulative score. Extension of
the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment.
[0167] In one preferred approach, the "percentage of sequence
identity" is determined by comparing two optimally aligned
sequences over a window of comparison of at least 20 positions,
wherein the portion of the polypeptide sequence in the comparison
window may comprise additions or deletions (ie., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical amino acid residue occurs in both sequences
to yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the reference
sequence (i.e., the window size) and multiplying the results by 100
to yield the percentage of sequence identity.
[0168] Within other illustrative embodiments, a polypeptide may be
a xenogeneic polypeptide that comprises an polypeptide having
substantial sequence identity, as described above, to the human
polypeptide (also termed autologous antigen) which served as a
reference polypeptide, but which xenogeneic polypeptide is derived
from a different, non-human species. One skilled in the art will
recognize that "self" antigens are often poor stimulators of CD8+
and CD4+ T-lymphocyte responses, and therefore efficient
immunotherapeutic strategies directed against tumor polypeptides
require the development of methods to overcome immune tolerance to
particular self tumor polypeptides. For example, humans immunized
with prostase protein from a xenogeneic (non human) origin are
capable of mounting an immune response against the counterpart
human protein, e.g. the human prostase tumor protein present on
human tumor cells. Accordingly, the present invention provides
methods for purifying the xenogeneic form of the tumor proteins set
forth herein, such as the polypeptides set forth in SEQ ID
NO:36-41, 56, 57, 61, 62, 92 and 96, or those encoded by
polynucleotide sequences set forth in SEQ ID NO: 1-35, 42-55,
58-60, 63-91 and 93-95.
[0169] Therefore, one aspect of the present invention provides
xenogeneic variants of the polypeptide compositions described
herein. Such xenogeneic variants generally encompassed by the
present invention will typically exhibit at least about 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or
more identity along their lengths, to a polypeptide sequences set
forth herein.
[0170] More particularly, the invention is directed to mouse, rat,
monkey, porcine and other non-human polypeptides which can be used
as xenogeneic forms of human polypeptides set forth herein, to
induce immune responses directed against tumor polypeptides of the
invention.
[0171] Within other illustrative embodiments, a polypeptide may be
a fusion polypeptide that comprises multiple polypeptides as
described herein, or that comprises at least one polypeptide as
described herein and an unrelated sequence, such as a known tumor
protein. A fusion partner may, for example, assist in providing T
helper epitopes (an immunological fusion partner), preferably T
helper epitopes recognized by humans, or may assist in expressing
the protein (an expression enhancer) at higher yields than the
native recombinant protein. Certain preferred fusion partners are
both immunological and expression enhancing fusion partners. Other
fusion partners may be selected so as to increase the solubility of
the polypeptide or to enable the polypeptide to be targeted to
desired intracellular compartments. Still further fusion partners
include affinity tags, which facilitate purification of the
polypeptide.
[0172] Fusion polypeptides may generally be prepared using standard
techniques, including chemical conjugation. Preferably, a fusion
polypeptide is expressed as a recombinant polypeptide, allowing the
production of increased levels, relative to a non-fused
polypeptide, in an expression system. Briefly, DNA sequences
encoding the polypeptide components may be assembled separately,
and ligated into an appropriate expression vector. The 3' end of
the DNA sequence encoding one polypeptide component is ligated,
with or without a peptide linker, to the 5' end of a DNA sequence
encoding the second polypeptide component so that the reading
frames of the sequences are in phase. This permits translation into
a single fusion polypeptide that retains the biological activity of
both component polypeptides.
[0173] A peptide linker sequence may be employed to separate the
first and second polypeptide components by a distance sufficient to
ensure that each polypeptide folds into its secondary and tertiary
structures. Such a peptide linker sequence is incorporated into the
fusion polypeptide using standard techniques well known in the art.
Suitable peptide linker sequences may be chosen based on the
following factors: (1) their ability to adopt a flexible extended
conformation; (2) their inability to adopt a secondary structure
that could interact with functional epitopes on the first and
second polypeptides; and (3) the lack of hydrophobic or charged
residues that might react with the polypeptide functional epitopes.
Preferred peptide linker sequences contain Gly, Asn and Ser
residues. Other near neutral amino acids, such as Thr and Ala may
also be used in the linker sequence. Amino acid sequences which may
be usefully employed as linkers include those disclosed in Maratea
et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci.
USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No.
4,751,180. The linker sequence may generally be from 1 to about 50
amino acids in length. Linker sequences are not required when the
first and second polypeptides have non-essential N-terminal amino
acid regions that can be used to separate the functional domains
and prevent steric interference.
[0174] The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The
regulatory elements responsible for expression of DNA are located
only 5' to the DNA sequence encoding the first polypeptides.
Similarly, stop codons required to end translation and
transcription termination signals are only present 3' to the DNA
sequence encoding the second polypeptide.
[0175] The fusion polypeptide can comprise a polypeptide as
described herein together with an unrelated immunogenic protein,
such as an immunogenic protein capable of eliciting a recall
response. Examples of such proteins include tetanus, tuberculosis
and hepatitis proteins (see, for example, Stoute et al. New Engl. J
Med., 336:86-91, 1997).
[0176] In one preferred embodiment, the immunological fusion
partner is derived from a Mycobacterium sp., such as a
Mycobacterium tuberculosis-derived Ral2 fragment. Ral2 compositions
and methods for their use in enhancing the expression and/or
immunogenicity of heterologous polynucleotide/polypeptide sequences
is described in U.S. patent application Ser. No. 60/158,585, the
disclosure of which is incorporated herein by reference in its
entirety. Briefly, Ral2 refers to a polynucleotide region that is a
subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid.
MTB32A is a serine protease of 32 KD molecular weight encoded by a
gene in virulent and avirulent strains of M. tuberculosis. The
nucleotide sequence and amino acid sequence of MTB32A have been
described (for example, U.S. patent application Ser. No.
60/158,585; see also, Skeiky et al., Infection and Immun. (1999)
67:3998-4007, incorporated herein by reference). C-terminal
fragments of the MTB32A coding sequence express at high levels and
remain as a soluble polypeptides throughout the purification
process. Moreover, Ral2 may enhance the immunogenicity of
heterologous immunogenic polypeptides with which it is fused. One
preferred Ral2 fusion polypeptide comprises a 14 KD C-terminal
fragment corresponding to amino acid residues 192 to 323 of MTB32A.
Other preferred Ral2 polynucleotides generally comprise at least
about 15 consecutive nucleotides, at least about 30 nucleotides, at
least about 60 nucleotides, at least about 100 nucleotides, at
least about 200 nucleotides, or at least about 300 nucleotides that
encode a portion of a Ral2 polypeptide. Ral2 polynucleotides may
comprise a native sequence (i.e., an endogenous sequence that
encodes a Ral2 polypeptide or a portion thereof) or may comprise a
variant of such a sequence. Ral2 polynucleotide variants may
contain one or more substitutions, additions, deletions and/or
insertions such that the biological activity of the encoded fusion
polypeptide is not substantially diminished, relative to a fusion
polypeptide comprising a native Ral2 polypeptide. Variants
preferably exhibit at least about 70% identity, more preferably at
least about 80% identity and most preferably at least about 90%
identity to a polynucleotide sequence that encodes a native Ral2
polypeptide or a portion thereof.
[0177] Within other preferred embodiments, an immunological fusion
partner is derived from protein D, a surface protein of the
gram-negative bacterium Haemophilus influenza B (WO 91/18926).
Preferably, a protein D derivative comprises approximately the
first third of the protein (e.g., the first N-terminal 100-110
amino acids), and a protein D derivative may be lipidated. Within
certain preferred embodiments, the first 109 residues of a
Lipoprotein D fusion partner is included on the N-terminus to
provide the polypeptide with additional exogenous T-cell epitopes
and to increase the expression level in E. coli (thus functioning
as an expression enhancer). The lipid tail ensures optimal
presentation of the antigen to antigen presenting cells. Other
fusion partners include the non-structural protein from influenzae
virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids
are used, although different fragments that include T-helper
epitopes may be used.
[0178] In another embodiment, the immunological fusion partner is
the protein known as LYTA, or a portion thereof (preferably a
C-terminal portion). LYTA is derived from Streptococcus pneumoniae,
which synthesizes an N-acetyl-L-alanine amidase known as amidase
LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an
autolysin that specifically degrades certain bonds in the
peptidoglycan backbone. The C-terminal domain of the LYTA protein
is responsible for the affinity to the choline or to some choline
analogues such as DEAE. This property has been exploited for the
development of E. coli C-LYTA expressing plasmids useful for
expression of fusion proteins. Purification of hybrid proteins
containing the C-LYTA fragment at the amino terminus has been
described (see Biotechnology 10:795-798, 1992). Within a preferred
embodiment, a repeat portion of LYTA may be incorporated into a
fusion polypeptide. A repeat portion is found in the C-terminal
region starting at residue 178. A particularly preferred repeat
portion incorporates residues 188-305.
[0179] Yet another illustrative embodiment involves fusion
polypeptides, and the polynucleotides encoding them, wherein the
fusion partner comprises a targeting signal capable of directing a
polypeptide to the endosomal/lysosomal compartment, as described in
U.S. Pat. No. 5,633,234. An immunogenic polypeptide of the
invention, when fused with this targeting signal, will associate
more efficiently with MHC class II molecules and thereby provide
enhanced in vivo stimulation of CD4.sup.+ T-cells specific for the
polypeptide.
[0180] Polypeptides of the invention are prepared using any of a
variety of well known synthetic and/or recombinant techniques, the
latter of which are further described below. Polypeptides, portions
and other variants generally less than about 150 amino acids can be
generated by synthetic means, using techniques well known to those
of ordinary skill in the art. In one illustrative example, such
polypeptides are synthesized using any of the commercially
available solid-phase techniques, such as the Merrifield
solid-phase synthesis method, where amino acids are sequentially
added to a growing amino acid chain. See Merrifield, J. Am. Chem.
Soc. 85:2149-2146, 1963. Equipment for automated synthesis of
polypeptides is commercially available from suppliers such as
Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and
may be operated according to the manufacturer's instructions.
[0181] In general, polypeptide compositions (including fusion
polypeptides) of the invention are isolated. An "isolated"
polypeptide is one that is removed from its original environment.
For example, a naturally-occurring protein or polypeptide is
isolated if it is separated from some or all of the coexisting
materials in the natural system. Preferably, such polypeptides are
also purified, e.g., are at least about 90% pure, more preferably
at least about 95% pure and most preferably at least about 99%
pure.
[0182] Polynucleotide Compositions
[0183] The present invention, in other aspects, provides
polynucleotide compositions. The terms "DNA" and "polynucleotide"
are used essentially interchangeably herein to refer to a DNA
molecule that has been isolated free of total genomic DNA of a
particular species. "Isolated," as used herein, means that a
polynucleotide is substantially away from other coding sequences,
and that the DNA molecule does not contain large portions of
unrelated coding DNA, such as large chromosomal fragments or other
functional genes or polypeptide coding regions. Of course, this
refers to the DNA molecule as originally isolated, and does not
exclude genes or coding regions later added to the segment by the
hand of man.
[0184] As will be understood by those skilled in the art, the
polynucleotide compositions of this invention can include genomic
sequences, extra-genomic and plasmid-encoded sequences and smaller
engineered gene segments that express, or may be adapted to
express, proteins, polypeptides, peptides and the like. Such
segments may be naturally isolated, or modified synthetically by
the hand of man.
[0185] As will be also recognized by the skilled artisan,
polynucleotides of the invention may be single-stranded (coding or
antisense) or double-stranded, and may be DNA (genomic, cDNA or
synthetic) or RNA molecules. RNA molecules may include HnRNA
molecules, which contain introns and correspond to a DNA molecule
in a one-to-one manner, and mRNA molecules, which do not contain
introns. Additional coding or non-coding sequences may, but need
not, be present within a polynucleotide of the present invention,
and a polynucleotide may, but need not, be linked to other
molecules and/or support materials.
[0186] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes a polypeptide/protein of the
invention or a portion thereof) or may comprise a sequence that
encodes a variant or derivative, preferably and immunogenic variant
or derivative, of such a sequence.
[0187] Therefore, according to another aspect of the present
invention, polynucleotide compositions are provided that comprise
some or all of a polynucleotide sequence set forth in any one of
SEQ ID NO:1-35, 42-55, 58-60, 63-91 and 93-95, complements of a
polynucleotide sequence set forth in any one of SEQ ID NO: 1-35,
42-55, 58-60, 63-91 and 93-95, and degenerate variants of a
polynucleotide sequence set forth in any one of SEQ ID NO:1-35,
42-55, 58-60, 63-91 and 93-95. In certain preferred embodiments,
the polynucleotide sequences set forth herein encode immunogenic
polypeptides, as described above.
[0188] In other related embodiments, the present invention provides
polynucleotide variants having substantial identity to the
sequences disclosed herein in SEQ ID NO:1-35, 42-55, 58-60, 63-91
and 93-95, for example those comprising at least 70% sequence
identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% or higher, sequence identity compared to a
polynucleotide sequence of this invention using the methods
described herein, (e.g., BLAST analysis using standard parameters,
as described below). One skilled in this art will recognize that
these values can be appropriately adjusted to determine
corresponding identity of proteins encoded by two nucleotide
sequences by taking into account codon degeneracy, amino acid
similarity, reading frame positioning and the like.
[0189] Typically, polynucleotide variants will contain one or more
substitutions, additions, deletions and/or insertions, preferably
such that the immunogenicity of the polypeptide encoded by the
variant polynucleotide is not substantially diminished relative to
a polypeptide encoded by a polynucleotide sequence specifically set
forth herein). The term "variants" should also be understood to
encompasses homologous genes of xenogenic origin.
[0190] In additional embodiments, the present invention provides
polynucleotide fragments comprising or consisting of various
lengths of contiguous stretches of sequence identical to or
complementary to one or more of the sequences disclosed herein. For
example, polynucleotides are provided by this invention that
comprise or consist of at least about 10, 15, 20, 30, 40, 50, 75,
100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides
of one or more of the sequences disclosed herein as well as all
intermediate lengths there between. It will be readily understood
that "intermediate lengths", in this context, means any length
between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22,
23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102,
103, etc.; 150, 151, 152, 153, etc.; including all integers through
200-500; 500-1,000, and the like. A polynucleotide sequence as
described here may be extended at one or both ends by additional
nucleotides not found in the native sequence. This additional
sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 nucleotides at either end of the
disclosed sequence or at both ends of the disclosed sequence.
[0191] In another embodiment of the invention, polynucleotide
compositions are provided that are capable of hybridizing under
moderate to high stringency conditions to a polynucleotide sequence
provided herein, or a fragment thereof, or a complementary sequence
thereof. Hybridization techniques are well known in the art of
molecular biology. For purposes of illustration, suitable
moderately stringent conditions for testing the hybridization of a
polynucleotide of this invention with other polynucleotides include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-60.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times.and 0.2.times.SSC
containing 0.1% SDS. One skilled in the art will understand that
the stringency of hybridization can be readily manipulated, such as
by altering the salt content of the hybridization solution and/or
the temperature at which the hybridization is performed. For
example, in another embodiment, suitable highly stringent
hybridization conditions include those described above, with the
exception that the temperature of hybridization is increased, e.g.,
to 60-65.degree. C. or 65-70.degree. C.
[0192] In certain preferred embodiments, the polynucleotides
described above, e.g., polynucleotide variants, fragments and
hybridizing sequences, encode polypeptides that are immunologically
cross-reactive with a polypeptide sequence specifically set forth
herein. In other preferred embodiments, such polynucleotides encode
polypeptides that have a level of immunogenic activity of at least
about 50%, preferably at least about 70%, and more preferably at
least about 90% of that for a polypeptide sequence specifically set
forth herein.
[0193] The polynucleotides of the present invention, or fragments
thereof, regardless of the length of the coding sequence itself,
may be combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol. For example, illustrative polynucleotide segments with
total lengths of about 10,000, about 5000, about 3000, about 2,000,
about 1,000, about 500, about 200, about 100, about 50 base pairs
in length, and the like, (including all intermediate lengths) are
contemplated to be useful in many implementations of this
invention.
[0194] When comparing polynucleotide sequences, two sequences are
said to be "identical" if the sequence of nucleotides in the two
sequences is the same when aligned for maximum correspondence, as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, 40 to about
50, in which a sequence may be compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned.
[0195] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E.
W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971)
Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
[0196] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0197] One preferred example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0
can be used, for example with the parameters described herein, to
determine percent sequence identity for the polynucleotides of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology
Information. In one illustrative example, cumulative scores can be
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, and expectation (E) of 10,
and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50,
expectation (E) of 10, M=5, N=-4 and a comparison of both
strands.
[0198] Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) of 20 percent or less,
usually 5 to 15 percent, or 10 to 12 percent, as compared to the
reference sequences (which does not comprise additions or
deletions) for optimal alignment of the two sequences. The
percentage is calculated by determining the number of positions at
which the identical nucleic acid bases occurs in both sequences to
yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the reference
sequence (i.e., the window size) and multiplying the results by 100
to yield the percentage of sequence identity.
[0199] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless,
polynucleotides that vary due to differences in codon usage are
specifically contemplated by the present invention. Further,
alleles of the genes comprising the polynucleotide sequences
provided herein are within the scope of the present invention.
Alleles are endogenous genes that are altered as a result of one or
more mutations, such as deletions, additions and/or substitutions
of nucleotides. The resulting mRNA and protein may, but need not,
have an altered structure or function. Alleles may be identified
using standard techniques (such as hybridization, amplification
and/or database sequence comparison).
[0200] Therefore, in another embodiment of the invention, a
mutagenesis approach, such as site-specific mutagenesis, is
employed for the preparation of immunogenic variants and/or
derivatives of the polypeptides described herein. By this approach,
specific modifications in a polypeptide sequence can be made
through mutagenesis of the underlying polynucleotides that encode
them. These techniques provides a straightforward approach to
prepare and test sequence variants, for example, incorporating one
or more of the foregoing considerations, by introducing one or more
nucleotide sequence changes into the polynucleotide.
[0201] Site-specific mutagenesis allows the production of mutants
through the use of specific oligonucleotide sequences which encode
the DNA sequence of the desired mutation, as well as a sufficient
number of adjacent nucleotides, to provide a primer sequence of
sufficient size and sequence complexity to form a stable duplex on
both sides of the deletion junction being traversed. Mutations may
be employed in a selected polynucleotide sequence to improve,
alter, decrease, modify, or otherwise change the properties of the
polynucleotide itself, and/or alter the properties, activity,
composition, stability, or primary sequence of the encoded
polypeptide.
[0202] In certain embodiments of the present invention, the
inventors contemplate the mutagenesis of the disclosed
polynucleotide sequences to alter one or more properties of the
encoded polypeptide, such as the immunogenicity of a polypeptide
vaccine. The techniques of site-specific mutagenesis are well-known
in the art, and are widely used to create variants of both
polypeptides and polynucleotides. For example, site-specific
mutagenesis is often used to alter a specific portion of a DNA
molecule. In such embodiments, a primer comprising typically about
14 to about 25 nucleotides or so in length is employed, with about
5 to about 10 residues on both sides of the junction of the
sequence being altered.
[0203] As will be appreciated by those of skill in the art,
site-specific mutagenesis techniques have often employed a phage
vector that exists in both a single stranded and double stranded
form. Typical vectors useful in site-directed mutagenesis include
vectors such as the M13 phage. These phage are readily
commercially-available and their use is generally well-known to
those skilled in the art. Double-stranded plasmids are also
routinely employed in site directed mutagenesis that eliminates the
step of transferring the gene of interest from a plasmid to a
phage.
[0204] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart of two strands of a double-stranded vector that includes
within its sequence a DNA sequence that encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are
selected which include recombinant vectors bearing the mutated
sequence arrangement.
[0205] The preparation of sequence variants of the selected
peptide-encoding DNA segments using site-directed mutagenesis
provides a means of producing potentially useful species and is not
meant to be limiting as there are other ways in which sequence
variants of peptides and the DNA sequences encoding them may be
obtained. For example, recombinant vectors encoding the desired
peptide sequence may be treated with mutagenic agents, such as
hydroxylamine, to obtain sequence variants. Specific details
regarding these methods and protocols are found in the teachings of
Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby,
1994; and Maniatis et al., 1982, each incorporated herein by
reference, for that purpose.
[0206] As used herein, the term "oligonucleotide directed
mutagenesis procedure" refers to template-dependent processes and
vector-mediated propagation which result in an increase in the
concentration of a specific nucleic acid molecule relative to its
initial concentration, or in an increase in the concentration of a
detectable signal, such as amplification. As used herein, the term
"oligonucleotide directed mutagenesis procedure" is intended to
refer to a process that involves the template-dependent extension
of a primer molecule. The term template dependent process refers to
nucleic acid synthesis of a RNA or a DNA molecule wherein the
sequence of the newly synthesized strand of nucleic acid is
dictated by the well-known rules of complementary base pairing
(see, for example, Watson, 1987). Typically, vector mediated
methodologies involve the introduction of the nucleic acid fragment
into a DNA or RNA vector, the clonal amplification of the vector,
and the recovery of the amplified nucleic acid fragment. Examples
of such methodologies are provided by U.S. Pat. No. 4,237,224,
specifically incorporated herein by reference in its entirety.
[0207] In another approach for the production of polypeptide
variants of the present invention, recursive sequence
recombination, as described in U.S. Pat. No. 5,837,458, may be
employed. In this approach, iterative cycles of recombination and
screening or selection are performed to "evolve" individual
polynucleotide variants of the invention having, for example,
enhanced immunogenic activity.
[0208] In other embodiments of the present invention, the
polynucleotide sequences provided herein can be advantageously used
as probes or primers for nucleic acid hybridization. As such, it is
contemplated that nucleic acid segments that comprise or consist of
a sequence region of at least about a 15 nucleotide long contiguous
sequence that has the same sequence as, or is complementary to, a
15 nucleotide long contiguous sequence disclosed herein will find
particular utility. Longer contiguous identical or complementary
sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000
(including all intermediate lengths) and even up to full length
sequences will also be of use in certain embodiments.
[0209] The ability of such nucleic acid probes to specifically
hybridize to a sequence of interest will enable them to be of use
in detecting the presence of complementary sequences in a given
sample. However, other uses are also envisioned, such as the use of
the sequence information for the preparation of mutant species
primers, or primers for use in preparing other genetic
constructions.
[0210] Polynucleotide molecules having sequence regions consisting
of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even
of 100-200 nucleotides or so (including intermediate lengths as
well), identical or complementary to a polynucleotide sequence
disclosed herein, are particularly contemplated as hybridization
probes for use in, e.g., Southern and Northern blotting. This would
allow a gene product, or fragment thereof, to be analyzed, both in
diverse cell types and also in various bacterial cells. The total
size of fragment, as well as the size of the complementary
stretch(es), will ultimately depend on the intended use or
application of the particular nucleic acid segment. Smaller
fragments will generally find use in hybridization embodiments,
wherein the length of the contiguous complementary region may be
varied, such as between about 15 and about 100 nucleotides, but
larger contiguous complementarity stretches may be used, according
to the length complementary sequences one wishes to detect.
[0211] The use of a hybridization probe of about 15-25 nucleotides
in length allows the formation of a duplex molecule that is both
stable and selective. Molecules having contiguous complementary
sequences over stretches greater than 15 bases in length are
generally preferred, though, in order to increase stability and
selectivity of the hybrid, and thereby improve the quality and
degree of specific hybrid molecules obtained. One will generally
prefer to design nucleic acid molecules having gene-complementary
stretches of 15 to 25 contiguous nucleotides, or even longer where
desired.
[0212] Hybridization probes may be selected from any portion of any
of the sequences disclosed herein. All that is required is to
review the sequences set forth herein, or to any continuous portion
of the sequences, from about 15-25 nucleotides in length up to and
including the full length sequence, that one wishes to utilize as a
probe or primer. The choice of probe and primer sequences may be
governed by various factors. For example, one may wish to employ
primers from towards the termini of the total sequence.
[0213] Small polynucleotide segments or fragments may be readily
prepared by, for example, directly synthesizing the fragment by
chemical means, as is commonly practiced using an automated
oligonucleotide synthesizer. Also, fragments may be obtained by
application of nucleic acid reproduction technology, such as the
PCR.TM. technology of U.S. Pat. No. 4,683,202 (incorporated herein
by reference), by introducing selected sequences into recombinant
vectors for recombinant production, and by other recombinant DNA
techniques generally known to those of skill in the art of
molecular biology.
[0214] The nucleotide sequences of the invention may be used for
their ability to selectively form duplex molecules with
complementary stretches of the entire gene or gene fragments of
interest. Depending on the application envisioned, one will
typically desire to employ varying conditions of hybridization to
achieve varying degrees of selectivity of probe towards target
sequence. For applications requiring high selectivity, one will
typically desire to employ relatively stringent conditions to form
the hybrids, e.g., one will select relatively low salt and/or high
temperature conditions, such as provided by a salt concentration of
from about 0.02 M to about 0.15 M salt at temperatures of from
about 50.degree. C. to about 70.degree. C. Such selective
conditions tolerate little, if any, mismatch between the probe and
the template or target strand, and would be particularly suitable
for isolating related sequences.
[0215] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template, less stringent (reduced
stringency) hybridization conditions will typically be needed in
order to allow formation of the heteroduplex. In these
circumstances, one may desire to employ salt conditions such as
those of from about 0.15 M to about 0.9 M salt, at temperatures
ranging from about 20.degree. C. to about 55.degree. C.
Cross-hybridizing species can thereby be readily identified as
positively hybridizing signals with respect to control
hybridizations. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0216] According to another embodiment of the present invention,
polynucleotide compositions comprising antisense oligonucleotides
are provided. Antisense oligonucleotides have been demonstrated to
be effective and targeted inhibitors of protein synthesis, and,
consequently, provide a therapeutic approach by which a disease can
be treated by inhibiting the synthesis of proteins that contribute
to the disease. The efficacy of antisense oligonucleotides for
inhibiting protein synthesis is well established. For example, the
synthesis of polygalactauronase and the muscarine type 2
acetylcholine receptor are inhibited by antisense oligonucleotides
directed to their respective mRNA sequences (U.S. Pat. No.
5,739,119 and U.S. Pat. No. 5,759,829). Further, examples of
antisense inhibition have been demonstrated with the nuclear
protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1,
E-selectin, STK-1, striatal GABA.sub.A receptor and human EGF
(Jaskulski et al., Science. 1988 Jun 10;240(4858):1544-6;
Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et
al., Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U.S. Pat.
No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and
U.S. Pat. No. 5,610,288). Antisense constructs have also been
described that inhibit and can be used to treat a variety of
abnormal cellular proliferations, e.g. cancer (U.S. Pat. No.
5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.
5,783,683).
[0217] Therefore, in certain embodiments, the present invention
provides oligonucleotide sequences that comprise all, or a portion
of, any sequence that is capable of specifically binding to
polynucleotide sequence described herein, or a complement thereof.
In one embodiment, the antisense oligonucleotides comprise DNA or
derivatives thereof. In another embodiment, the oligonucleotides
comprise RNA or derivatives thereof. In a third embodiment, the
oligonucleotides are modified DNAs comprising a phosphorothioated
modified backbone. In a fourth embodiment, the oligonucleotide
sequences comprise peptide nucleic acids or derivatives thereof. In
each case, preferred compositions comprise a sequence region that
is complementary, and more preferably substantially-complementary,
and even more preferably, completely complementary to one or more
portions of polynucleotides disclosed herein. Selection of
antisense compositions specific for a given gene sequence is based
upon analysis of the chosen target sequence and determination of
secondary structure, Tm, binding energy, and relative stability.
Antisense compositions may be selected based upon their relative
inability to form dimers, hairpins, or other secondary structures
that would reduce or prohibit specific binding to the target mRNA
in a host cell. Highly preferred target regions of the mRNA, are
those which are at or near the AUG translation initiation codon,
and those sequences which are substantially complementary to 5'
regions of the mRNA. These secondary structure analyses and target
site selection considerations can be performed, for example, using
v.4 of the OLIGO primer analysis software and/or the BLASTN 2.0.5
algorithm software (Altschul et al., Nucleic Acids Res. 1997,
25(17):3389-402).
[0218] The use of an antisense delivery method employing a short
peptide vector, termed MPG (27 residues), is also contemplated. The
MPG peptide contains a hydrophobic domain derived from the fusion
sequence of HIV gp41 and a hydrophilic domain from the nuclear
localization sequence of SV40 T-antigen (Morris et al., Nucleic
Acids Res. 1997 Jul 15;25(14):2730-6). It has been demonstrated
that several molecules of the MPG peptide coat the antisense
oligonucleotides and can be delivered into cultured mammalian cells
in less than 1 hour with relatively high efficiency (90%). Further,
the interaction with MPG strongly increases both the stability of
the oligonucleotide to nuclease and the ability to cross the plasma
membrane.
[0219] According to another embodiment of the invention, the
polynucleotide compositions described herein are used in the design
and preparation of ribozyme molecules for inhibiting expression of
the tumor polypeptides and proteins of the present invention in
tumor cells. Ribozymes are RNA-protein complexes that cleave
nucleic acids in a site-specific fashion. Ribozymes have specific
catalytic domains that possess endonuclease activity (Kim and Cech,
Proc Natl Acad Sci U S A. 1987 Dec;84(24):8788-92; Forster and
Symons, Cell. 1987 Apr 24;49(2):211-20). For example, a large
number of ribozymes accelerate phosphoester transfer reactions with
a high degree of specificity, often cleaving only one of several
phosphoesters in an oligonucleotide substrate (Cech et al., Cell.
1981 Dec;27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990
Dec 5;216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May
14;357(6374):173-6). This specificity has been attributed to the
requirement that the substrate bind via specific base-pairing
interactions to the internal guide sequence ("IGS") of the ribozyme
prior to chemical reaction.
[0220] Six basic varieties of naturally-occurring enzymatic RNAs
are known presently. Each can catalyze the hydrolysis of RNA
phosphodiester bonds in trans (and thus can cleave other RNA
molecules) under physiological conditions. In general, enzymatic
nucleic acids act by first binding to a target RNA. Such binding
occurs through the target binding portion of a enzymatic nucleic
acid which is held in close proximity to an enzymatic portion of
the molecule that acts to cleave the target RNA. Thus, the
enzymatic nucleic acid first recognizes and then binds a target RNA
through complementary base-pairing, and once bound to the correct
site, acts enzymatically to cut the target RNA. Strategic cleavage
of such a target RNA will destroy its ability to direct synthesis
of an encoded protein. After an enzymatic nucleic acid has bound
and cleaved its RNA target, it is released from that RNA to search
for another target and can repeatedly bind and cleave new
targets.
[0221] The enzymatic nature of a ribozyme is advantageous over many
technologies, such as antisense technology (where a nucleic acid
molecule simply binds to a nucleic acid target to block its
translation) since the concentration of ribozyme necessary to
affect a therapeutic treatment is lower than that of an antisense
oligonucleotide. This advantage reflects the ability of the
ribozyme to act enzymatically. Thus, a single ribozyme molecule is
able to cleave many molecules of target RNA. In addition, the
ribozyme is a highly specific inhibitor, with the specificity of
inhibition depending not only on the base pairing mechanism of
binding to the target RNA, but also on the mechanism of target RNA
cleavage. Single mismatches, or base-substitutions, near the site
of cleavage can completely eliminate catalytic activity of a
ribozyme. Similar mismatches in antisense molecules do not prevent
their action (Woolf et al., Proc Natl Acad Sci U S A. 1992 Aug
15;89(16):7305-9). Thus, the specificity of action of a ribozyme is
greater than that of an antisense oligonucleotide binding the same
RNA site.
[0222] The enzymatic nucleic acid molecule may be formed in a
hammerhead, hairpin, a hepatitis .delta. virus, group I intron or
RNaseP RNA (in association with an RNA guide sequence) or
Neurospora VS RNA motif. Examples of hammerhead motifs are
described by Rossi et al. Nucleic Acids Res. 1992 Sep
11;20(17):4559-65. Examples of hairpin motifs are described by
Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and
Tritz, Biochemistry 1989 Jun 13;28(12):4929-33; Hampel et al.,
Nucleic Acids Res. 1990 Jan 25;18(2):299-304 and U.S. Pat. No.
5,631,359. An example of the hepatitis .delta. virus motif is
described by Perrotta and Been, Biochemistry. 1992 Dec
1;31(47):11843-52; an example of the RNaseP motif is described by
Guerrier-Takada et al., Cell. 1983 Dec;35(3 Pt 2):849-57;
Neurospora VS RNA ribozyme motif is described by Collins (Saville
and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins,
Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8826-30; Collins and
Olive, Biochemistry. 1993 Mar 23;32(11):2795-9); and an example of
the Group I intron is described in (U.S. Pat. No. 4,987,071). All
that is important in an enzymatic nucleic acid molecule of this
invention is that it has a specific substrate binding site which is
complementary to one or more of the target gene RNA regions, and
that it have nucleotide sequences within or surrounding that
substrate binding site which impart an RNA cleaving activity to the
molecule. Thus the ribozyme constructs need not be limited to
specific motifs mentioned herein.
[0223] Ribozymes may be designed as described in Int. Pat. Appl.
Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595,
each specifically incorporated herein by reference) and synthesized
to be tested in vitro and in vivo, as described. Such ribozymes can
also be optimized for delivery. While specific examples are
provided, those in the art will recognize that equivalent RNA
targets in other species can be utilized when necessary.
[0224] Ribozyme activity can be optimized by altering the length of
the ribozyme binding arms, or chemically synthesizing ribozymes
with modifications that prevent their degradation by serum
ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065;
Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO
91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No.
5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which
describe various chemical modifications that can be made to the
sugar moieties of enzymatic RNA molecules), modifications which
enhance their efficacy in cells, and removal of stem II bases to
shorten RNA synthesis times and reduce chemical requirements.
[0225] Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595)
describes the general methods for delivery of enzymatic RNA
molecules. Ribozymes may be administered to cells by a variety of
methods known to those familiar to the art, including, but not
restricted to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive
microspheres. For some indications, ribozymes may be directly
delivered ex vivo to cells or tissues with or without the
aforementioned vehicles. Alternatively, the RNA/vehicle combination
may be locally delivered by direct inhalation, by direct injection
or by use of a catheter, infusion pump or stent. Other routes of
delivery include, but are not limited to, intravascular,
intramuscular, subcutaneous or joint injection, aerosol inhalation,
oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. More detailed
descriptions of ribozyme delivery and administration are provided
in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ.
No. WO 93/23569, each specifically incorporated herein by
reference.
[0226] Another means of accumulating high concentrations of a
ribozyme(s) within cells is to incorporate the ribozyme-encoding
sequences into a DNA expression vector. Transcription of the
ribozyme sequences are driven from a promoter for eukaryotic RNA
polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase
III (pol III). Transcripts from pol II or pol III promoters will be
expressed at high levels in all cells; the levels of a given pol II
promoter in a given cell type will depend on the nature of the gene
regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic RNA polymerase promoters may also be used, providing
that the prokaryotic RNA polymerase enzyme is expressed in the
appropriate cells Ribozymes expressed from such promoters have been
shown to function in mammalian cells. Such transcription units can
be incorporated into a variety of vectors for introduction into
mammalian cells, including but not restricted to, plasmid DNA
vectors, viral DNA vectors (such as adenovirus or adeno-associated
vectors), or viral RNA vectors (such as retroviral, semliki forest
virus, sindbis virus vectors).
[0227] In another embodiment of the invention, peptide nucleic
acids (PNAs) compositions are provided. PNA is a DNA mimic in which
the nucleobases are attached to a pseudopeptide backbone (Good and
Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is
able to be utilized in a number methods that traditionally have
used RNA or DNA. Often PNA sequences perform better in techniques
than the corresponding RNA or DNA sequences and have utilities that
are not inherent to RNA or DNA. A review of PNA including methods
of making, characteristics of, and methods of using, is provided by
Corey (Trends Biotechnol 1997 Jun;15(6):224-9). As such, in certain
embodiments, one may prepare PNA sequences that are complementary
to one or more portions of the ACE mRNA sequence, and such PNA
compositions may be used to regulate, alter, decrease, or reduce
the translation of ACE-specific mRNA, and thereby alter the level
of ACE activity in a host cell to which such PNA compositions have
been administered.
[0228] PNAs have 2-aminoethyl-glycine linkages replacing the normal
phosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec
6;254(5037):1497-500; Hanvey et al., Science. 1992 Nov
27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996
Jan;4(1):5-23). This chemistry has three important consequences:
firstly, in contrast to DNA or phosphorothioate oligonucleotides,
PNAs are neutral molecules; secondly, PNAs are achiral, which
avoids the need to develop a stereoselective synthesis; and
thirdly, PNA synthesis uses standard Boc or Fmoc protocols for
solid-phase peptide synthesis, although other methods, including a
modified Merrifield method, have been used.
[0229] PNA monomers or ready-made oligomers are commercially
available from PerSeptive Biosystems (Framingham, Mass.). PNA
syntheses by either Boc or Fmoc protocols are straightforward using
manual or automated protocols (Norton et al., Bioorg Med Chem. 1995
Apr;3(4):437-45). The manual protocol lends itself to the
production of chemically modified PNAs or the simultaneous
synthesis of families of closely related PNAs.
[0230] As with peptide synthesis, the success of a particular PNA
synthesis will depend on the properties of the chosen sequence. For
example, while in theory PNAs can incorporate any combination of
nucleotide bases, the presence of adjacent purines can lead to
deletions of one or more residues in the product. In expectation of
this difficulty, it is suggested that, in producing PNAs with
adjacent purines, one should repeat the coupling of residues likely
to be added inefficiently. This should be followed by the
purification of PNAs by reverse-phase high-pressure liquid
chromatography, providing yields and purity of product similar to
those observed during the synthesis of peptides.
[0231] Modifications of PNAs for a given application may be
accomplished by coupling amino acids during solid-phase synthesis
or by attaching compounds that contain a carboxylic acid group to
the exposed N-terminal amine. Alternatively, PNAs can be modified
after synthesis by coupling to an introduced lysine or cysteine.
The ease with which PNAs can be modified facilitates optimization
for better solubility or for specific functional requirements. Once
synthesized, the identity of PNAs and their derivatives can be
confirmed by mass spectrometry. Several studies have made and
utilized modifications of PNAs (for example, Norton et al., Bioorg
Med Chem. 1995 Apr;3(4):437-45; Petersen et al., J Pept Sci. 1995
May-Jun;1(3):175-83; Orum et al., Biotechniques. 1995
Sep;19(3):472-80; Footer et al., Biochemistry. 1996 Aug
20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug
11;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci U S A. 1995
Jun 6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci U S A. 1995
Mar 14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug
15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci U S A. 1997
Nov 11;94(23):12320-5; Seeger et al., Biotechniques. 1997
Sep;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA
chimeric molecules and their uses in diagnostics, modulating
protein in organisms, and treatment of conditions susceptible to
therapeutics.
[0232] Methods of characterizing the antisense binding properties
of PNAs are discussed in Rose (Anal Chem. 1993 Dec
15;65(24):3545-9) and Jensen et al. (Biochemistry. 1997 Apr
22;36(16):5072-7). Rose uses capillary gel electrophoresis to
determine binding of PNAs to their complementary oligonucleotide,
measuring the relative binding kinetics and stoichiometry. Similar
types of measurements were made by Jensen et al. using BIAcore.TM.
technology.
[0233] Other applications of PNAs that have been described and will
be apparent to the skilled artisan include use in DNA strand
invasion, antisense inhibition, mutational analysis, enhancers of
transcription, nucleic acid purification, isolation of
transcriptionally active genes, blocking of transcription factor
binding, genome cleavage, biosensors, in situ hybridization, and
the like.
[0234] Polynucleotide Identification, Characterization and
Expression
[0235] Polynucleotides compositions of the present invention may be
identified, prepared and/or manipulated using any of a variety of
well established techniques (see generally, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratories, Cold Spring Harbor, N.Y., 1989, and other like
references). For example, a polynucleotide may be identified, as
described in more detail below, by screening a microarray of cDNAs
for tumor-associated expression (i.e., expression that is at least
two fold greater in a tumor than in normal tissue, as determined
using a representative assay provided herein). Such screens may be
performed, for example, using the microarray technology of
Affymetrix, Inc. (Santa Clara, Calif.) according to the
manufacturer's instructions (and essentially as described by Schena
et al., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller
et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997).
Alternatively, polynucleotides may be amplified from cDNA prepared
from cells expressing the proteins described herein, such as tumor
cells.
[0236] Many template dependent processes are available to amplify a
target sequences of interest present in a sample. One of the best
known amplification methods is the polymerase chain reaction
(PCR.TM.) which is described in detail in U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159, each of which is incorporated herein by
reference in its entirety. Briefly, in PCR.TM., two primer
sequences are prepared which are complementary to regions on
opposite complementary strands of the target sequence. An excess of
deoxynucleoside triphosphates is added to a reaction mixture along
with a DNA polymerase (e.g., Taq polymerase). If the target
sequence is present in a sample, the primers will bind to the
target and the polymerase will cause the primers to be extended
along the target sequence by adding on nucleotides. By raising and
lowering the temperature of the reaction mixture, the extended
primers will dissociate from the target to form reaction products,
excess primers will bind to the target and to the reaction product
and the process is repeated. Preferably reverse transcription and
PCR.TM. amplification procedure may be performed in order to
quantify the amount of mRNA amplified. Polymerase chain reaction
methodologies are well known in the art.
[0237] Any of a number of other template dependent processes, many
of which are variations of the PCR.TM. amplification technique, are
readily known and available in the art. Illustratively, some such
methods include the ligase chain reaction (referred to as LCR),
described, for example, in Eur. Pat. Appl. Publ. No. 320,308 and
U.S. Pat. No. 4,883,750; Qbeta Replicase, described in PCT Intl.
Pat. Appl. Publ. No. PCT/US87/00880; Strand Displacement
Amplification (SDA) and Repair Chain Reaction (RCR). Still other
amplification methods are described in Great Britain Pat. Appl. No.
2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025.
Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS) (PCT Intl. Pat.
Appl. Publ. No. WO 88/10315), including nucleic acid sequence based
amplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822
describes a nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO
89/06700 describes a nucleic acid sequence amplification scheme
based on the hybridization of a promoter/primer sequence to a
target single-stranded DNA ("ssDNA") followed by transcription of
many RNA copies of the sequence. Other amplification methods such
as "RACE" (Frohman, 1990), and "one-sided PCR" (Ohara, 1989) are
also well-known to those of skill in the art.
[0238] An amplified portion of a polynucleotide of the present
invention may be used to isolate a full length gene from a suitable
library (e.g., a tumor cDNA library) using well known techniques.
Within such techniques, a library (cDNA or genomic) is screened
using one or more polynucleotide probes or primers suitable for
amplification. Preferably, a library is size-selected to include
larger molecules. Random primed libraries may also be preferred for
identifying 5' and upstream regions of genes. Genomic libraries are
preferred for obtaining introns and extending 5' sequences.
[0239] For hybridization techniques, a partial sequence may be
labeled (e.g., by nick-translation or end-labeling with .sup.32P)
using well known techniques. A bacterial or bacteriophage library
is then generally screened by hybridizing filters containing
denatured bacterial colonies (or lawns containing phage plaques)
with the labeled probe (see Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring
Harbor, N.Y., 1989). Hybridizing colonies or plaques are selected
and expanded, and the DNA is isolated for further analysis. cDNA
clones may be analyzed to determine the amount of additional
sequence by, for example, PCR using a primer from the partial
sequence and a primer from the vector. Restriction maps and partial
sequences may be generated to identify one or more overlapping
clones. The complete sequence may then be determined using standard
techniques, which may involve generating a series of deletion
clones. The resulting overlapping sequences can then assembled into
a single contiguous sequence. A full length cDNA molecule can be
generated by ligating suitable fragments, using well known
techniques.
[0240] Alternatively, amplification techniques, such as those
described above, can be useful for obtaining a full length coding
sequence from a partial cDNA sequence. One such amplification
technique is inverse PCR (see Triglia et al., Nucl. Acids Res.
16:8186, 1988), which uses restriction enzymes to generate a
fragment in the known region of the gene. The fragment is then
circularized by intramolecular ligation and used as a template for
PCR with divergent primers derived from the known region. Within an
alternative approach, sequences adjacent to a partial sequence may
be retrieved by amplification with a primer to a linker sequence
and a primer specific to a known region. The amplified sequences
are typically subjected to a second round of amplification with the
same linker primer and a second primer specific to the known
region. A variation on this procedure, which employs two primers
that initiate extension in opposite directions from the known
sequence, is described in WO 96/38591. Another such technique is
known as "rapid amplification of cDNA ends" or RACE. This technique
involves the use of an internal primer and an external primer,
which hybridizes to a polyA region or vector sequence, to identify
sequences that are 5' and 3' of a known sequence. Additional
techniques include capture PCR (Lagerstrom et al., PCR Methods
Applic. 1:111-19, 1991) and walking PCR (Parker et al., Nucl.
Acids. Res. 19:3055-60, 1991). Other methods employing
amplification may also be employed to obtain a full length cDNA
sequence.
[0241] In certain instances, it is possible to obtain a full length
cDNA sequence by analysis of sequences provided in an expressed
sequence tag (EST) database, such as that available from GenBank.
Searches for overlapping ESTs may generally be performed using well
known programs (e.g., NCBI BLAST searches), and such ESTs may be
used to generate a contiguous full length sequence. Full length DNA
sequences may also be obtained by analysis of genomic
fragments.
[0242] In other embodiments of the invention, polynucleotide
sequences or fragments thereof which encode polypeptides of the
invention, or fusion proteins or functional equivalents thereof,
may be used in recombinant DNA molecules to direct expression of a
polypeptide in appropriate host cells. Due to the inherent
degeneracy of the genetic code, other DNA sequences that encode
substantially the same or a functionally equivalent amino acid
sequence may be produced and these sequences may be used to clone
and express a given polypeptide.
[0243] As will be understood by those of skill in the art, it may
be advantageous in some instances to produce polypeptide-encoding
nucleotide sequences possessing non-naturally occurring codons. For
example, codons preferred by a particular prokaryotic or eukaryotic
host can be selected to increase the rate of protein expression or
to produce a recombinant RNA transcript having desirable
properties, such as a half-life which is longer than that of a
transcript generated from the naturally occurring sequence.
[0244] Moreover, the polynucleotide sequences of the present
invention can be engineered using methods generally known in the
art in order to alter polypeptide encoding sequences for a variety
of reasons, including but not limited to, alterations which modify
the cloning, processing, and/or expression of the gene product. For
example, DNA shuffling by random fragmentation and PCR reassembly
of gene fragments and synthetic oligonucleotides may be used to
engineer the nucleotide sequences. In addition, site-directed
mutagenesis may be used to insert new restriction sites, alter
glycosylation patterns, change codon preference, produce splice
variants, or introduce mutations, and so forth.
[0245] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences may be ligated to a
heterologous sequence to encode a fusion protein. For example, to
screen peptide libraries for inhibitors of polypeptide activity, it
may be useful to encode a chimeric protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the
polypeptide-encoding sequence and the heterologous protein
sequence, so that the polypeptide may be cleaved and purified away
from the heterologous moiety.
[0246] Sequences encoding a desired polypeptide may be synthesized,
in whole or in part, using chemical methods well known in the art
(see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.
215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.
225-232). Alternatively, the protein itself may be produced using
chemical methods to synthesize the amino acid sequence of a
polypeptide, or a portion thereof. For example, peptide synthesis
can be performed using various solid-phase techniques (Roberge, J.
Y. et al. (1995) Science 269:202-204) and automated synthesis may
be achieved, for example, using the ABI 431A Peptide Synthesizer
(Perkin Elmer, Palo Alto, Calif.).
[0247] A newly synthesized peptide may be substantially purified by
preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins, Structures and Molecular Principles,
WH Freeman and Co., New York, N.Y.) or other comparable techniques
available in the art. The composition of the synthetic peptides may
be confirmed by amino acid analysis or sequencing (e.g., the Edman
degradation procedure). Additionally, the amino acid sequence of a
polypeptide, or any part thereof, may be altered during direct
synthesis and/or combined using chemical methods with sequences
from other proteins, or any part thereof, to produce a variant
polypeptide.
[0248] In order to express a desired polypeptide, the nucleotide
sequences encoding the polypeptide, or functional equivalents, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods which are well
known to those skilled in the art may be used to construct
expression vectors containing sequences encoding a polypeptide of
interest and appropriate transcriptional and translational control
elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described, for example, in
Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et
al. (1989) Current Protocols in Molecular Biology, John Wiley &
Sons, New York. N.Y.
[0249] A variety of expression vector/host systems may be utilized
to contain and express polynucleotide sequences. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems.
[0250] The "control elements" or "regulatory sequences" present in
an expression vector are those non-translated regions of the
vector--enhancers, promoters, 5' and 3' untranslated regions--which
interact with host cellular proteins to carry out transcription and
translation. Such elements may vary in their strength and
specificity. Depending on the vector system and host utilized, any
number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used. For
example, when cloning in bacterial systems, inducible promoters
such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid
(Stratagene, La Jolla, Calif.) or pSPORT1 plasmid (Gibco BRL,
Gaithersburg, Md.) and the like may be used. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are generally preferred. If it is necessary to generate a cell line
that contains multiple copies of the sequence encoding a
polypeptide, vectors based on SV40 or EBV may be advantageously
used with an appropriate selectable marker.
[0251] In bacterial systems, any of a number of expression vectors
may be selected depending upon the use intended for the expressed
polypeptide. For example, when large quantities are needed, for
example for the induction of antibodies, vectors which direct high
level expression of fusion proteins that are readily purified may
be used. Such vectors include, but are not limited to, the
multifunctional E. coli cloning and expression vectors such as
pBLUESCRIPT (Stratagene), in which the sequence encoding the
polypeptide of interest may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,
Madison, Wis.) may also be used to express foreign polypeptides as
fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems may be designed to include heparin, thrombin, or factor XA
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0252] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol.
153:516-544.
[0253] In cases where plant expression vectors are used, the
expression of sequences encoding polypeptides may be driven by any
of a number of promoters. For example, viral promoters such as the
35S and 19S promoters of CaMV may be used alone or in combination
with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311. Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196).
[0254] An insect system may also be used to express a polypeptide
of interest. For example, in one such system, Autographa
califormica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes in Spodoptera frugiperda cells or in
Trichoplusia larvae. The sequences encoding the polypeptide may be
cloned into a non-essential region of the virus, such as the
polyhedrin gene, and placed under control of the polyhedrin
promoter. Successful insertion of the polypeptide-encoding sequence
will render the polyhedrin gene inactive and produce recombinant
virus lacking coat protein. The recombinant viruses may then be
used to infect, for example, S. frugiperda cells or Trichoplusia
larvae in which the polypeptide of interest may be expressed
(Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. 91
:3224-3227).
[0255] In mammalian host cells, a number of viral-based expression
systems are generally available. For example, in cases where an
adenovirus is used as an expression vector, sequences encoding a
polypeptide of interest may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter
and tripartite leader sequence. Insertion in a non-essential E1 or
E3 region of the viral genome may be used to obtain a viable virus
which is capable of expressing the polypeptide in infected host
cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci.
81:3655-3659). In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells.
[0256] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding a polypeptide of
interest. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding the
polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous translational control signals including the
ATG initiation codon should be provided. Furthermore, the
initiation codon should be in the correct reading frame to ensure
translation of the entire insert. Exogenous translational elements
and initiation codons may be of various origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers which are appropriate for the particular
cell system which is used, such as those described in the
literature (Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162).
[0257] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation. glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells such as CHO, COS, HeLa, MDCK, HEK293, and W138, which
have specific cellular machinery and characteristic mechanisms for
such post-translational activities, may be chosen to ensure the
correct modification and processing of the foreign protein.
[0258] For long-term, high-yield production of recombinant
proteins, stable expression is generally preferred. For example,
cell lines which stably express a polynucleotide of interest may be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0259] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1990) Cell 22:817-23) genes which can be employed in tk.sup.-
or aprt.sup.-cells, respectively. Also, antimetabolite, antibiotic
or herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad Sci. 77:3567-70); npt, which confers
resistance to the aminoglycosides, neomycin and G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-51). The use of visible markers has gained popularity with
such markers as anthocyanins, beta-glucuronidase and its substrate
GUS, and luciferase and its substrate luciferin, being widely used
not only to identify transformants, but also to quantify the amount
of transient or stable protein expression attributable to a
specific vector system (Rhodes, C. A. et al. (1995) Methods Mol.
Biol. 55:121-131).
[0260] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. For example, if the
sequence encoding a polypeptide is inserted within a marker gene
sequence, recombinant cells containing sequences can be identified
by the absence of marker gene function. Alternatively, a marker
gene can be placed in tandem with a polypeptide-encoding sequence
under the control of a single promoter. Expression of the marker
gene in response to induction or selection usually indicates
expression of the tandem gene as well.
[0261] Alternatively, host cells that contain and express a desired
polynucleotide sequence may be identified by a variety of
procedures known to those of skill in the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridizations
and protein bioassay or immunoassay techniques which include, for
example, membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein.
[0262] A variety of protocols for detecting and measuring the
expression of polynucleotide-encoded products, using either
polyclonal or monoclonal antibodies specific for the product are
known in the art. Examples include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), and fluorescence activated
cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two non-interfering
epitopes on a given polypeptide may be preferred for some
applications, but a competitive binding assay may also be employed.
These and other assays are described, among other places, in
Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual,
APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J Exp.
Med. 158:1211-1216).
[0263] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides include oligolabeling, nick translation,
end-labeling or PCR amplification using a labeled nucleotide.
Alternatively, the sequences, or any portions thereof may be cloned
into a vector for the production of an mRNA probe. Such vectors are
known in the art, are commercially available, and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These
procedures may be conducted using a variety of commercially
available kits. Suitable reporter molecules or labels, which may be
used include radionuclides, enzymes, fluorescent, chemiluminescent,
or chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0264] Host cells transformed with a polynucleotide sequence of
interest may be cultured under conditions suitable for the
expression and recovery of the protein from cell culture. The
protein produced by a recombinant cell may be secreted or contained
intracellularly depending on the sequence and/or the vector used.
As will be understood by those of skill in the art, expression
vectors containing polynucleotides of the invention may be designed
to contain signal sequences which direct secretion of the encoded
polypeptide through a prokaryotic or eukaryotic cell membrane.
Other recombinant constructions may be used to join sequences
encoding a polypeptide of interest to nucleotide sequence encoding
a polypeptide domain which will facilitate purification of soluble
proteins. Such purification facilitating domains include, but are
not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences such as those
specific for Factor XA or enterokinase (Invitrogen. San Diego,
Calif.) between the purification domain and the encoded polypeptide
may be used to facilitate purification. One such expression vector
provides for expression of a fusion protein containing a
polypeptide of interest and a nucleic acid encoding 6 histidine
residues preceding a thioredoxin or an enterokinase cleavage site.
The histidine residues facilitate purification on IMIAC
(immobilized metal ion affinity chromatography) as described in
Porath, J. et al. (1992, Prot. Exp. Purif 3:263-281) while the
enterokinase cleavage site provides a means for purifying the
desired polypeptide from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
[0265] In addition to recombinant production methods, polypeptides
of the invention, and fragments thereof, may be produced by direct
peptide synthesis using solid-phase techniques (Merrifield J.
(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be
performed using manual techniques or by automation. Automated
synthesis may be achieved, for example, using Applied Biosystems
431A Peptide Synthesizer (Perkin Elmer). Alternatively, various
fragments may be chemically synthesized separately and combined
using chemical methods to produce the full length molecule.
[0266] Antibody Compositions Fragments Thereof and Other Binding
Agents
[0267] According to another aspect, the present invention further
provides binding agents, such as antibodies and antigen-binding
fragments thereof, that exhibit immunological binding to a tumor
polypeptide disclosed herein, or to a portion, variant or
derivative thereof. An antibody, or antigen-binding fragment
thereof, is said to "specifically bind," "immunogically bind,"
and/or is "immunologically reactive" to a polypeptide of the
invention if it reacts at a detectable level (within, for example,
an ELISA assay) with the polypeptide, and does not react detectably
with unrelated polypeptides under similar conditions.
[0268] Immunological binding, as used in this context, generally
refers to the non-covalent interactions of the type which occur
between an immunoglobulin molecule and an antigen for which the
immunoglobulin is specific. The strength, or affinity of
immunological binding interactions can be expressed in terms of the
dissociation constant (K.sub.d) of the interaction, wherein a
smaller K.sub.d represents a greater affinity. Immunological
binding properties of selected polypeptides can be quantified using
methods well known in the art. One such method entails measuring
the rates of antigen-binding site/antigen complex formation and
dissociation, wherein those rates depend on the concentrations of
the complex partners, the affinity of the interaction, and on
geometric parameters that equally influence the rate in both
directions. Thus, both the "on rate constant" (K.sub.on) and the
"off rate constant" (K.sub.off) can be determined by calculation of
the concentrations and the actual rates of association and
dissociation. The ratio of K.sub.off/K.sub.on enables cancellation
of all parameters not related to affinity, and is thus equal to the
dissociation constant K.sub.d. See, generally, Davies et al. (1990)
Annual Rev. Biochem. 59:439-473.
[0269] An "antigen-binding site," or "binding portion" of an
antibody refers to the part of the immunoglobulin molecule that
participates in antigen binding. The antigen binding site is formed
by amino acid residues of the N-terminal variable ("V") regions of
the heavy ("H") and light ("L") chains. Three highly divergent
stretches within the V regions of the heavy and light chains are
referred to as "hypervariable regions" which are interposed between
more conserved flanking stretches known as "framework regions," or
"FRs". Thus the term "FR" refers to amino acid sequences which are
naturally found between and adjacent to hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs."
[0270] Binding agents may be further capable of differentiating
between patients with and without a cancer, such as lung cancer,
using the representative assays provided herein. For example,
antibodies or other binding agents that bind to a tumor protein
will preferably generate a signal indicating the presence of a
cancer in at least about 20% of patients with the disease, more
preferably at least about 30% of patients. Alternatively, or in
addition, the antibody will generate a negative signal indicating
the absence of the disease in at least about 90% of individuals
without the cancer. To determine whether a binding agent satisfies
this requirement, biological samples (e.g., blood, sera, sputum,
urine and/or tumor biopsies) from patients with and without a
cancer (as determined using standard clinical tests) may be assayed
as described herein for the presence of polypeptides that bind to
the binding agent. Preferably, a statistically significant number
of samples with and without the disease will be assayed. Each
binding agent should satisfy the above criteria; however, those of
ordinary skill in the art will recognize that binding agents may be
used in combination to improve sensitivity.
[0271] Any agent that satisfies the above requirements may be a
binding agent. For example, a binding agent may be a ribosome, with
or without a peptide component, an RNA molecule or a polypeptide.
In a preferred embodiment, a binding agent is an antibody or an
antigen-binding fragment thereof. Antibodies may be prepared by any
of a variety of techniques known to those of ordinary skill in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988. In general, antibodies can be
produced by cell culture techniques, including the generation of
monoclonal antibodies as described herein, or via transfection of
antibody genes into suitable bacterial or mammalian cell hosts, in
order to allow for the production of recombinant antibodies. In one
technique, an immunogen comprising the polypeptide is initially
injected into any of a wide variety of mammals (e.g., mice, rats,
rabbits, sheep or goats). In this step, the polypeptides of this
invention may serve as the immunogen without modification.
Alternatively, particularly for relatively short polypeptides, a
superior immune response may be elicited if the polypeptide is
joined to a carrier protein, such as bovine serum albumin or
keyhole limpet hemocyanin. The immunogen is injected into the
animal host, preferably according to a predetermined schedule
incorporating one or more booster immunizations, and the animals
are bled periodically. Polyclonal antibodies specific for the
polypeptide may then be purified from such antisera by, for
example, affinity chromatography using the polypeptide coupled to a
suitable solid support.
[0272] Monoclonal antibodies specific for an antigenic polypeptide
of interest may be prepared, for example, using the technique of
Kohler and Milstein, Eur. J Immunol. 6:511-519, 1976, and
improvements thereto. Briefly, these methods involve the
preparation of immortal cell lines capable of producing antibodies
having the desired specificity (i.e., reactivity with the
polypeptide of interest). Such cell lines may be produced, for
example, from spleen cells obtained from an animal immunized as
described above. The spleen cells are then immortalized by, for
example, fusion with a myeloma cell fusion partner, preferably one
that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and
myeloma cells may be combined with a nonionic detergent for a few
minutes and then plated at low density on a selective medium that
supports the growth of hybrid cells, but not myeloma cells. A
preferred selection technique uses HAT (hypoxanthine, aminopterin,
thymidine) selection. After a sufficient time, usually about 1 to 2
weeks, colonies of hybrids are observed. Single colonies are
selected and their culture supernatants tested for binding activity
against the polypeptide. Hybridomas having high reactivity and
specificity are preferred.
[0273] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction. The
polypeptides of this invention may be used in the purification
process in, for example, an affinity chromatography step.
[0274] A number of therapeutically useful molecules are known in
the art which comprise antigen-binding sites that are capable of
exhibiting immunological binding properties of an antibody
molecule. The proteolytic enzyme papain preferentially cleaves IgG
molecules to yield several fragments, two of which (the "F(ab)"
fragments) each comprise a covalent heterodimer that includes an
intact antigen-binding site. The enzyme pepsin is able to cleave
IgG molecules to provide several fragments, including the
"F(ab').sub.2 " fragment which comprises both antigen-binding
sites. An "Fv" fragment can be produced by preferential proteolytic
cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin
molecule. Fv fragments are, however, more commonly derived using
recombinant techniques known in the art. The Fv fragment includes a
non-covalent V.sub.H::V.sub.L heterodimer including an
antigen-binding site which retains much of the antigen recognition
and binding capabilities of the native antibody molecule. Inbar et
al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al.
(1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem
19:4091-4096.
[0275] A single chain Fv ("sFv") polypeptide is a covalently linked
V.sub.H::V.sub.L heterodimer which is expressed from a gene fusion
including V.sub.H- and V.sub.L-encoding genes linked by a
peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci.
USA 85(16):5879-5883. A number of methods have been described to
discern chemical structures for converting the naturally
aggregated--but chemically separated--light and heavy polypeptide
chains from an antibody V region into an sFv molecule which will
fold into a three dimensional structure substantially similar to
the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.
5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No.
4,946,778, to Ladner et al.
[0276] Each of the above-described molecules includes a heavy chain
and a light chain CDR set, respectively interposed between a heavy
chain and a light chain FR set which provide support to the CDRS
and define the spatial relationship of the CDRs relative to each
other. As used herein, the term "CDR set" refers to the three
hypervariable regions of a heavy or light chain V region.
Proceeding from the N-terminus of a heavy or light chain, these
regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An
antigen-binding site, therefore, includes six CDRs, comprising the
CDR set from each of a heavy and a light chain V region. A
polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3)
is referred to herein as a "molecular recognition unit."
Crystallographic analysis of a number of antigen-antibody complexes
has demonstrated that the amino acid residues of CDRs form
extensive contact with bound antigen, wherein the most extensive
antigen contact is with the heavy chain CDR3. Thus, the molecular
recognition units are primarily responsible for the specificity of
an antigen-binding site.
[0277] As used herein, the term "FR set" refers to the four
flanking amino acid sequences which frame the CDRs of a CDR set of
a heavy or light chain V region. Some FR residues may contact bound
antigen; however, FRs are primarily responsible for folding the V
region into the antigen-binding site, particularly the FR residues
directly adjacent to the CDRS. Within FRs, certain amino residues
and certain structural features are very highly conserved. In this
regard, all V region sequences contain an internal disulfide loop
of around 90 amino acid residues. When the V regions fold into a
binding-site, the CDRs are displayed as projecting loop motifs
which form an antigen-binding surface. It is generally recognized
that there are conserved structural regions of FRs which influence
the folded shape of the CDR loops into certain "canonical"
structures--regardless of the precise CDR amino acid sequence.
Further, certain FR residues are known to participate in
non-covalent interdomain contacts which stabilize the interaction
of the antibody heavy and light chains.
[0278] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent V
regions and their associated CDRs fused to human constant domains
(Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989)
Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J
Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res.
47:3577-3583), rodent CDRs grafted into a human supporting FR prior
to fusion with an appropriate human antibody constant domain
(Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al.
(1988) Science 239:1534-1536; and Jones et al. (1986) Nature
321:522-525), and rodent CDRs supported by recombinantly veneered
rodent FRs (European Patent Publication No. 519,596, published Dec.
23, 1992). These "humanized" molecules are designed to minimize
unwanted immunological response toward rodent antihuman antibody
molecules which limits the duration and effectiveness of
therapeutic applications of those moieties in human recipients.
[0279] As used herein, the terms "veneered FRs" and "recombinantly
veneered FRs" refer to the selective replacement of FR residues
from, e.g., a rodent heavy or light chain V region, with human FR
residues in order to provide a xenogeneic molecule comprising an
antigen-binding site which retains substantially all of the native
FR polypeptide folding structure. Veneering techniques are based on
the understanding that the ligand binding characteristics of an
antigen-binding site are determined primarily by the structure and
relative disposition of the heavy and light chain CDR sets within
the antigen-binding surface. Davies et al. (1990) Ann. Rev.
Biochem. 59:439-473. Thus, antigen binding specificity can be
preserved in a humanized antibody only wherein the CDR structures,
their interaction with each other, and their interaction with the
rest of the V region domains are carefully maintained. By using
veneering techniques, exterior (e.g., solvent-accessible) FR
residues which are readily encountered by the immune system are
selectively replaced with human residues to provide a hybrid
molecule that comprises either a weakly immunogenic, or
substantially non-immunogenic veneered surface.
[0280] The process of veneering makes use of the available sequence
data for human antibody variable domains compiled by Kabat et al.,
in Sequences of Proteins of Immunological Interest, 4th ed., (U.S.
Dept. of Health and Human Services, U.S. Government Printing
Office, 1987), updates to the Kabat database, and other accessible
U.S. and foreign databases (both nucleic acid and protein). Solvent
accessibilities of V region amino acids can be deduced from the
known three-dimensional structure for human and murine antibody
fragments. There are two general steps in veneering a murine
antigen-binding site. Initially, the FRs of the variable domains of
an antibody molecule of interest are compared with corresponding FR
sequences of human variable domains obtained from the
above-identified sources. The most homologous human V regions are
then compared residue by residue to corresponding murine amino
acids. The residues in the murine FR which differ from the human
counterpart are replaced by the residues present in the human
moiety using recombinant techniques well known in the art. Residue
switching is only carried out with moieties which are at least
partially exposed (solvent accessible), and care is exercised in
the replacement of amino acid residues which may have a significant
effect on the tertiary structure of V region domains, such as
proline, glycine and charged amino acids.
[0281] In this manner, the resultant "veneered" murine
antigen-binding sites are thus designed to retain the murine CDR
residues, the residues substantially adjacent to the CDRs, the
residues identified as buried or mostly buried (solvent
inaccessible), the residues believed to participate in non-covalent
(e.g., electrostatic and hydrophobic) contacts between heavy and
light chain domains, and the residues from conserved structural
regions of the FRs which are believed to influence the "canonical"
tertiary structures of the CDR loops. These design criteria are
then used to prepare recombinant nucleotide sequences which combine
the CDRs of both the heavy and light chain of a murine
antigen-binding site into human-appearing FRs that can be used to
transfect mammalian cells for the expression of recombinant human
antibodies which exhibit the antigen specificity of the murine
antibody molecule.
[0282] In another embodiment of the invention, monoclonal
antibodies of the present invention may be coupled to one or more
therapeutic agents. Suitable agents in this regard include
radionuclides, differentiation inducers, drugs, toxins, and
derivatives thereof. Preferred radionuclides include .sup.90Y,
.sup.123I, .sup.125I, .sup.131I, .sup.186Re, .sup.188Re,
.sup.211At, and .sup.212Bi. Preferred drugs include methotrexate,
and pyrimidine and purine analogs. Preferred differentiation
inducers include phorbol esters and butyric acid. Preferred toxins
include ricin, abrin, diptheria toxin, cholera toxin, gelonin,
Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral
protein.
[0283] A therapeutic agent may be coupled (e.g., covalently bonded)
to a suitable monoclonal antibody either directly or indirectly
(e.g., via a linker group). A direct reaction between an agent and
an antibody is possible when each possesses a substituent capable
of reacting with the other. For example, a nucleophilic group, such
as an amino or sulfhydryl group, on one may be capable of reacting
with a carbonyl-containing group, such as an anhydride or an acid
halide, or with an alkyl group containing a good leaving group
(e.g., a halide) on the other.
[0284] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0285] It will be evident to those skilled in the art that a
variety of bifunctional or polyfunctional reagents, both homo- and
hetero-functional (such as those described in the catalog of the
Pierce Chemical Co., Rockford, Ill.), may be employed as the linker
group. Coupling may be effected, for example, through amino groups,
carboxyl groups, sulfhydryl groups or oxidized carbohydrate
residues. There are numerous references describing such
methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.
[0286] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,
U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,
to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,
U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).
[0287] It may be desirable to couple more than one agent to an
antibody. In one embodiment, multiple molecules of an agent are
coupled to one antibody molecule. In another embodiment, more than
one type of agent may be coupled to one antibody. Regardless of the
particular embodiment, immunoconjugates with more than one agent
may be prepared in a variety of ways. For example, more than one
agent may be coupled directly to an antibody molecule, or linkers
that provide multiple sites for attachment can be used.
Alternatively, a carrier can be used.
[0288] A carrier may bear the agents in a variety of ways,
including covalent bonding either directly or via a linker group.
Suitable carriers include proteins such as albumins (e.g., U.S.
Pat. No. 4,507,234, to Kato et al.), peptides and polysaccharides
such as aminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et
al.). A carrier may also bear an agent by noncovalent bonding or by
encapsulation, such as within a liposome vesicle (e.g., U.S. Pat.
Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide
agents include radiohalogenated small molecules and chelating
compounds. For example, U.S. Pat. No. 4,735,792 discloses
representative radiohalogenated small molecules and their
synthesis. A radionuclide chelate may be formed from chelating
compounds that include those containing nitrogen and sulfur atoms
as the donor atoms for binding the metal, or metal oxide,
radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et
al. discloses representative chelating compounds and their
synthesis.
[0289] T Cell Compositions
[0290] The present invention, in another aspect, provides T cells
specific for a tumor polypeptide disclosed herein, or for a variant
or derivative thereof. Such cells may generally be prepared in
vitro or ex vivo, using standard procedures. For example, T cells
may be isolated from bone marrow, peripheral blood, or a fraction
of bone marrow or peripheral blood of a patient, using a
commercially available cell separation system, such as the
Isolex.TM. System, available from Nexell Therapeutics, Inc.
(Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No.
5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).
Alternatively, T cells may be derived from related or unrelated
humans, non-human mammals, cell lines or cultures.
[0291] T cells may be stimulated with a polypeptide, polynucleotide
encoding a polypeptide and/or an antigen presenting cell (APC) that
expresses such a polypeptide. Such stimulation is performed under
conditions and for a time sufficient to permit the generation of T
cells that are specific for the polypeptide of interest.
Preferably, a tumor polypeptide or polynucleotide of the invention
is present within a delivery vehicle, such as a microsphere, to
facilitate the generation of specific T cells.
[0292] T cells are considered to be specific for a polypeptide of
the present invention if the T cells specifically proliferate,
secrete cytokines or kill target cells coated with the polypeptide
or expressing a gene encoding the polypeptide. T cell specificity
may be evaluated using any of a variety of standard techniques. For
example, within a chromium release assay or proliferation assay, a
stimulation index of more than two fold increase in lysis and/or
proliferation, compared to negative controls, indicates T cell
specificity. Such assays may be performed, for example, as
described in Chen et al., Cancer Res. 54:1065-1070, 1994.
Alternatively, detection of the proliferation of T cells may be
accomplished by a variety of known techniques. For example, T cell
proliferation can be detected by measuring an increased rate of DNA
synthesis (e.g., by pulse-labeling cultures of T cells with
tritiated thymidine and measuring the amount of tritiated thymidine
incorporated into DNA). Contact with a tumor polypeptide (100 ng/ml
-100 .mu.g/ml, preferably 200 ng/ml -25 .mu.g/ml) for 3-7 days will
typically result in at least a two fold increase in proliferation
of the T cells. Contact as described above for 2-3 hours should
result in activation of the T cells, as measured using standard
cytokine assays in which a two fold increase in the level of
cytokine release (e.g., TNF or IFN-.gamma.) is indicative of T cell
activation (see Coligan et al., Current Protocols in Immunology,
vol. 1, Wiley Interscience (Greene 1998)). T cells that have been
activated in response to a tumor polypeptide, polynucleotide or
polypeptide-expressing APC may be CD4.sup.+ and/or CD8.sup.+. Tumor
polypeptide-specific T cells may be expanded using standard
techniques. Within preferred embodiments, the T cells are derived
from a patient, a related donor or an unrelated donor, and are
administered to the patient following stimulation and
expansion.
[0293] For therapeutic purposes, CD4.sup.+ or CD8.sup.+ T cells
that proliferate in response to a tumor polypeptide, polynucleotide
or APC can be expanded in number either in vitro or in vivo.
Proliferation of such T cells in vitro may be accomplished in a
variety of ways. For example, the T cells can be re-exposed to a
tumor polypeptide, or a short peptide corresponding to an
immunogenic portion of such a polypeptide, with or without the
addition of T cell growth factors, such as interleukin-2, and/or
stimulator cells that synthesize a tumor polypeptide.
Alternatively, one or more T cells that proliferate in the presence
of the tumor polypeptide can be expanded in number by cloning.
Methods for cloning cells are well known in the art, and include
limiting dilution.
[0294] T Cell Receptor Compositions
[0295] The T cell receptor (TCR) consists of 2 different, highly
variable polypeptide chains, termed the T-cell receptor .alpha. and
.beta. chains, that are linked by a disulfide bond (Janeway,
Travers, Walport. Immunobiology. Fourth Ed., 148-159. Elsevier
Science Ltd/Garland Publishing. 1999). The .alpha./.beta.
heterodimer complexes with the invariant CD3 chains at the cell
membrane. This complex recognizes specific antigenic peptides bound
to MHC molecules. The enormous diversity of TCR specificities is
generated much like immunoglobulin diversity, through somatic gene
rearrangement. The .alpha. chain genes contain over 50 variable
(V), 2 diversity (D), over 10 joining (J) segments, and 2 constant
region segments (C). The a chain genes contain over 70 V segments,
and over 60 J segments but no D segments, as well as one C segment.
During T cell development in the thymus, the D to J gene
rearrangement of the .beta. chain occurs, followed by the V gene
segment rearrangement to the DJ. This functional VDJ.sub..beta.
exon is transcribed and spliced to join to a C.sub..beta.. For the
.alpha. chain, a V.sub..alpha. gene segment rearranges to a
J.sub..alpha. gene segment to create the functional exon that is
then transcribed and spliced to the C.sub..alpha.. Diversity is
further increased during the recombination process by the random
addition of P and N-nucleotides between the V, D, and J segments of
the .beta. chain and between the V and J segments in the a chain
(Janeway, Travers, Walport. Immunobiology. Fourth Ed., 98 and 150.
Elsevier Science Ltd/Garland Publishing. 1999).
[0296] The present invention, in another aspect, provides TCRs
specific for a polypeptide disclosed herein, or for a variant or
derivative thereof. In accordance with the present invention,
polynucleotide and amino acid sequences are provided for the V-J or
V-D-J junctional regions or parts thereof for the alpha and beta
chains of the T-cell receptor which recognize tumor polypeptides
described herein. In general, this aspect of the invention relates
to T-cell receptors which recognize or bind tumor polypeptides
presented in the context of MHC. In a preferred embodiment the
tumor antigens recognized by the T-cell receptors comprise a
polypeptide of the present invention. For example, cDNA encoding a
TCR specific for a lung tumor peptide can be isolated from T cells
specific for a tumor polypeptide using standard molecular
biological and recombinant DNA techniques.
[0297] This invention further includes the T-cell receptors or
analogs thereof having substantially the same function or activity
as the T-cell receptors of this invention which recognize or bind
tumor polypeptides. Such receptors include, but are not limited to,
a fragment of the receptor, or a substitution, addition or deletion
mutant of a T-cell receptor provided herein. This invention also
encompasses polypeptides or peptides that are substantially
homologous to the T-cell receptors provided herein or that retain
substantially the same activity. The term "analog" includes any
protein or polypeptide having an amino acid residue sequence
substantially identical to the T-cell receptors provided herein in
which one or more residues, preferably no more than 5 residues,
more preferably no more than 25 residues have been conservatively
substituted with a functionally similar residue and which displays
the functional aspects of the T-cell receptor as described
herein.
[0298] The present invention further provides for suitable
mammalian host cells, for example, non-specific T cells, that are
transfected with a polynucleotide encoding TCRs specific for a
polypeptide described herein, thereby rendering the host cell
specific for the polypeptide. The .alpha. and .beta. chains of the
TCR may be contained on separate expression vectors or
alternatively, on a single expression vector that also contains an
internal ribosome entry site (IRES) for cap-independent translation
of the gene downstream of the IRES. Said host cells expressing TCRs
specific for the polypeptide may be used, for example, for adoptive
immunotherapy of lung cancer as discussed further below.
[0299] In further aspects of the present invention, cloned TCRs
specific for a polypeptide recited herein may be used in a kit for
the diagnosis of lung cancer. For example, the nucleic acid
sequence or portions thereof, of tumor-specific TCRs can be used as
probes or primers for the detection of expression of the rearranged
genes encoding the specific TCR in a biological sample. Therefore,
the present invention further provides for an assay for detecting
messenger RNA or DNA encoding the TCR specific for a
polypeptide.
[0300] Pharmaceutical Compositions
[0301] In additional embodiments, the present invention concerns
formulation of one or more of the polynucleotide, polypeptide,
T-cell, TCR, and/or antibody compositions disclosed herein in
pharmaceutically-acceptable carriers for administration to a cell
or an animal, either alone, or in combination with one or more
other modalities of therapy.
[0302] It will be understood that, if desired, a composition as
disclosed herein may be administered in combination with other
agents as well, such as, e.g., other proteins or polypeptides or
various pharmaceutically-active agents. In fact, there is virtually
no limit to other components that may also be included, given that
the additional agents do not cause a significant adverse effect
upon contact with the target cells or host tissues. The
compositions may thus be delivered along with various other agents
as required in the particular instance. Such compositions may be
purified from host cells or other biological sources, or
alternatively may be chemically synthesized as described herein.
Likewise, such compositions may further comprise substituted or
derivatized RNA or DNA compositions.
[0303] Therefore, in another aspect of the present invention,
pharmaceutical compositions are provided comprising one or more of
the polynucleotide, polypeptide, antibody, TCR, and/or T-cell
compositions described herein in combination with a physiologically
acceptable carrier. In certain preferred embodiments, the
pharmaceutical compositions of the invention comprise immunogenic
polynucleotide and/or polypeptide compositions of the invention for
use in prophylactic and theraputic vaccine applications. Vaccine
preparation is generally described in, for example, M. F. Powell
and M. J. Newman, eds., "Vaccine Design (the subunit and adjuvant
approach)," Plenum Press (NY, 1995). Generally, such compositions
will comprise one or more polynucleotide and/or polypeptide
compositions of the present invention in combination with one or
more immunostimulants.
[0304] It will be apparent that any of the pharmaceutical
compositions described herein can contain pharmaceutically
acceptable salts of the polynucleotides and polypeptides of the
invention. Such salts can be prepared, for example, from
pharmaceutically acceptable non-toxic bases, including organic
bases (e.g., salts of primary, secondary and tertiary amines and
basic amino acids) and inorganic bases (e.g., sodium, potassium,
lithium, ammonium, calcium and magnesium salts).
[0305] In another embodiment, illustrative immunogenic
compositions, e.g., vaccine compositions, of the present invention
comprise DNA encoding one or more of the polypeptides as described
above, such that the polypeptide is generated in situ. As noted
above, the polynucleotide may be administered within any of a
variety of delivery systems known to those of ordinary skill in the
art. Indeed, numerous gene delivery techniques are well known in
the art, such as those described by Rolland, Crit. Rev. Therap.
Drug Carrier Systems 15:143-198, 1998, and references cited
therein. Appropriate polynucleotide expression systems will, of
course, contain the necessary regulatory DNA regulatory sequences
for expression in a patient (such as a suitable promoter and
terminating signal). Alternatively, bacterial delivery systems may
involve the administration of a bacterium (such as
Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of
the polypeptide on its cell surface or secretes such an
epitope.
[0306] Therefore, in certain embodiments, polynucleotides encoding
immunogenic polypeptides described herein are introduced into
suitable mammalian host cells for expression using any of a number
of known viral-based systems. In one illustrative embodiment,
retroviruses provide a convenient and effective platform for gene
delivery systems. A selected nucleotide sequence encoding a
polypeptide of the present invention can be inserted into a vector
and packaged in retroviral particles using techniques known in the
art. The recombinant virus can then be isolated and delivered to a
subject. A number of illustrative retroviral systems have been
described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989)
BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy
1:5-14; Scarpa et al. (1991) Virology 180:849-852; Bums et al.
(1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie
and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
[0307] In addition, a number of illustrative adenovirus-based
systems have also been described. Unlike retroviruses which
integrate into the host genome, adenoviruses persist
extrachromosomally thus minimizing the risks associated with
insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol.
57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder
et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J.
Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;
Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al.
(1993) Human Gene Therapy 4:461-476).
[0308] Various adeno-associated virus (AAV) vector systems have
also been developed for polynucleotide delivery. AAV vectors can be
readily constructed using techniques well known in the art. See,
e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International
Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al.
(1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990)
Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J.
(1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N.
(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin,
R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith
(1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med.
179:1867-1875.
[0309] Additional viral vectors useful for delivering the
polynucleotides encoding polypeptides of the present invention by
gene transfer include those derived from the pox family of viruses,
such as vaccinia virus and avian poxvirus. By way of example,
vaccinia virus recombinants expressing the novel molecules can be
constructed as follows. The DNA encoding a polypeptide is first
inserted into an appropriate vector so that it is adjacent to a
vaccinia promoter and flanking vaccinia DNA sequences, such as the
sequence encoding thymidine kinase (TK). This vector is then used
to transfect cells which are simultaneously infected with vaccinia.
Homologous recombination serves to insert the vaccinia promoter
plus the gene encoding the polypeptide of interest into the viral
genome. The resulting TK.sup.(-) recombinant can be selected by
culturing the cells in the presence of 5-bromodeoxyuridine and
picking viral plaques resistant thereto.
[0310] A vaccinia-based infection/transfection system can be
conveniently used to provide for inducible, transient expression or
coexpression of one or more polypeptides described herein in host
cells of an organism. In this particular system, cells are first
infected in vitro with a vaccinia virus recombinant that encodes
the bacteriophage T7 RNA polymerase. This polymerase displays
exquisite specificity in that it only transcribes templates bearing
T7 promoters. Following infection, cells are transfected with the
polynucleotide or polynucleotides of interest, driven by a T7
promoter. The polymerase expressed in the cytoplasm from the
vaccinia virus recombinant transcribes the transfected DNA into RNA
which is then translated into polypeptide by the host translational
machinery. The method provides for high level, transient,
cytoplasmic production of large quantities of RNA and its
translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl.
Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad.
Sci. USA (1986) 83:8122-8126.
[0311] Alternatively, avipoxviruses, such as the fowlpox and
canarypox viruses, can also be used to deliver the coding sequences
of interest. Recombinant avipox viruses, expressing immunogens from
mammalian pathogens, are known to confer protective immunity when
administered to non-avian species. The use of an Avipox vector is
particularly desirable in human and other mammalian species since
members of the Avipox genus can only productively replicate in
susceptible avian species and therefore are not infective in
mammalian cells. Methods for producing recombinant Avipoxviruses
are known in the art and employ genetic recombination, as described
above with respect to the production of vaccinia viruses. See,
e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
[0312] Any of a number of alphavirus vectors can also be used for
delivery of polynucleotide compositions of the present invention,
such as those vectors described in U.S. Pat. Nos. 5,843,723;
6,015,686; 6,008,035 and 6,015,694. Certain vectors based on
Venezuelan Equine Encephalitis (VEE) can also be used, illustrative
examples of which can be found in U.S. Pat. Nos. 5,505,947 and
5,643,576.
[0313] Moreover, molecular conjugate vectors, such as the
adenovirus chimeric vectors described in Michael et al. J. Biol.
Chem. (1993) 268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci.
USA (1992) 89:6099-6103, can also be used for gene delivery under
the invention.
[0314] Additional illustrative information on these and other known
viral-based delivery systems can be found, for example, in
Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989;
Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et
al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330,
and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651;
EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988;
Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc.
Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc.
Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al.,
Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res.
73:1202-1207, 1993.
[0315] In certain embodiments, a polynucleotide may be integrated
into the genome of a target cell. This integration may be in the
specific location and orientation via homologous recombination
(gene replacement) or it may be integrated in a random,
non-specific location (gene augmentation). In yet further
embodiments, the polynucleotide may be stably maintained in the
cell as a separate, episomal segment of DNA. Such polynucleotide
segments or "episomes" encode sequences sufficient to permit
maintenance and replication independent of or in synchronization
with the host cell cycle. The manner in which the expression
construct is delivered to a cell and where in the cell the
polynucleotide remains is dependent on the type of expression
construct employed.
[0316] In another embodiment of the invention, a polynucleotide is
administered/delivered as "naked" DNA, for example as described in
Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen,
Science 259:1691-1692, 1993. The uptake of naked DNA may be
increased by coating the DNA onto biodegradable beads, which are
efficiently transported into the cells.
[0317] In still another embodiment, a composition of the present
invention can be delivered via a particle bombardment approach,
many of which have been described. In one illustrative example,
gas-driven particle acceleration can be achieved with devices such
as those manufactured by Powderject Pharmaceuticals PLC (Oxford,
UK) and Powderject Vaccines Inc. (Madison, Wis.), some examples of
which are described in U.S. Pat. Nos. 5,846,796; 6,010,478;
5,865,796; 5,584,807; and EP Patent No. 0500 799. This approach
offers a needle-free delivery approach wherein a dry powder
formulation of microscopic particles, such as polynucleotide or
polypeptide particles, are accelerated to high speed within a
helium gas jet generated by a hand held device, propelling the
particles into a target tissue of interest.
[0318] In a related embodiment, other devices and methods that may
be useful for gas-driven needle-less injection of compositions of
the present invention include those provided by Bioject, Inc.
(Portland, Oreg.), some examples of which are described in U.S.
Pat. Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163;
5,520,639 and 5,993,412.
[0319] According to another embodiment, the pharmaceutical
compositions described herein will comprise one or more
immunostimulants in addition to the immunogenic polynucleotide,
polypeptide, antibody, T-cell, TCR, and/or APC compositions of this
invention. An immunostimulant refers to essentially any substance
that enhances or potentiates an immune response (antibody and/or
cell-mediated) to an exogenous antigen. One preferred type of
immunostimulant comprises an adjuvant. Many adjuvants contain a
substance designed to protect the antigen from rapid catabolism,
such as aluminum hydroxide or mineral oil, and a stimulator of
immune responses, such as lipid A, Bortadella pertussis or
Mycobacterium tuberculosis derived proteins. Certain adjuvants are
commercially available as, for example, Freund's Incomplete
Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,
Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);
AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such
as aluminum hydroxide gel (alum) or aluminum phosphate; salts of
calcium, iron or zinc; an insoluble suspension of acylated
tyrosine; acylated sugars; cationically or anionically derivatized
polysaccharides; polyphosphazenes; biodegradable microspheres;
monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF,
interleukin-2,-7,-12, and other like growth factors, may also be
used as adjuvants.
[0320] Within certain embodiments of the invention, the adjuvant
composition is preferably one that induces an immune response
predominantly of the Th1 type. High levels of Th1-type cytokines
(e.g., IFN-.gamma., TNF.alpha., IL-2 and IL-12) tend to favor the
induction of cell mediated immune responses to an administered
antigen. In contrast, high levels of Th2-type cytokines (e.g.,
IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral
immune responses. Following application of a vaccine as provided
herein, a patient will support an immune response that includes
Th1- and Th2-type responses. Within a preferred embodiment, in
which a response is predominantly Th1-type, the level of Th1-type
cytokines will increase to a greater extent than the level of
Th2-type cytokines. The levels of these cytokines may be readily
assessed using standard assays. For a review of the families of
cytokines, see Mosmann and Coffinan, Ann. Rev. Immunol. 7:145-173,
1989.
[0321] Certain preferred adjuvants for eliciting a predominantly
Th1-type response include, for example, a combination of
monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl
lipid A, together with an aluminum salt. MPL.RTM. adjuvants are
available from Corixa Corporation (Seattle, Wash.; see, for
example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and
4,912,094). CpG-containing oligonucleotides (in which the CpG
dinucleotide is unmethylated) also induce a predominantly Th1
response. Such oligonucleotides are well known and are described,
for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos.
6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also
described, for example, by Sato et al., Science 273:352, 1996.
Another preferred adjuvant comprises a saponin, such as Quil A, or
derivatives thereof, including QS21 and QS7 (Aquila
Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or
Gypsophila or Chenopodium quinoa saponins . Other preferred
formulations include more than one saponin in the adjuvant
combinations of the present invention, for example combinations of
at least two of the following group comprising QS21, QS7, Quil A,
.beta.-escin, or digitonin.
[0322] Alternatively the saponin formulations may be combined with
vaccine vehicles composed of chitosan or other polycationic
polymers, polylactide and polylactide-co-glycolide particles,
poly-N-acetyl glucosamine-based polymer matrix, particles composed
of polysaccharides or chemically modified polysaccharides,
liposomes and lipid-based particles, particles composed of glycerol
monoesters, etc. The saponins may also be formulated in the
presence of cholesterol to form particulate structures such as
liposomes or ISCOMs. Furthermore, the saponins may be formulated
together with a polyoxyethylene ether or ester, in either a
non-particulate solution or suspension, or in a particulate
structure such as a paucilamelar liposome or ISCOM. The saponins
may also be formulated with excipients such as Carbopol.sup.R to
increase viscosity, or may be formulated in a dry powder form with
a powder excipient such as lactose.
[0323] In one preferred embodiment, the adjuvant system includes
the combination of a monophosphoryl lipid A and a saponin
derivative, such as the combination of QS21 and 3D-MPL.RTM.
adjuvant, as described in WO 94/00153, or a less reactogenic
composition where the QS21 is quenched with cholesterol, as
described in WO 96/33739. Other preferred formulations comprise an
oil-in-water emulsion and tocopherol. Another particularly
preferred adjuvant formulation employing QS21, 3D-MPL.RTM. adjuvant
and tocopherol in an oil-in-water emulsion is described in WO
95/17210.
[0324] Another enhanced adjuvant system involves the combination of
a CpG-containing oligonucleotide and a saponin derivative
particularly the combination of CpG and QS21 is disclosed in WO
00/09159. Preferably the formulation additionally comprises an oil
in water emulsion and tocopherol.
[0325] Additional illustrative adjuvants for use in the
pharmaceutical compositions of the invention include Montanide ISA
720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS
(CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2
or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium),
Detox (Enhanzyn.RTM.) (Corixa, Hamilton, Mont.), RC-529 (Corixa,
Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates
(AGPs), such as those described in pending U.S. patent application
Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are
incorporated herein by reference in their entireties, and
polyoxyethylene ether adjuvants such as those described in WO
99/52549A1.
[0326] Other preferred adjuvants include adjuvant molecules of the
general formula
(I): HO(CH.sub.2CH.sub.2O).sub.n--A--R,
[0327] wherein, n is 1-50, A is a bond or --C(O)--, R is C.sub.1-50
alkyl or Phenyl C.sub.1-50 alkyl.
[0328] One embodiment of the present invention consists of a
vaccine formulation comprising a polyoxyethylene ether of general
formula (I), wherein n is between 1 and 50, preferably 4-24, most
preferably 9; the R component is C.sub.1-50, preferably
C.sub.4-C.sub.20 alkyl and most preferably C.sub.12 alkyl, and A is
a bond. The concentration of the polyoxyethylene ethers should be
in the range 0.1-20%, preferably from 0.1-10%, and most preferably
in the range 0.1-1%. Preferred polyoxyethylene ethers are selected
from the following group: polyoxyethylene-9-lauryl ether,
polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,
polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,
and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as
polyoxyethylene lauryl ether are described in the Merck index
(12.sup.th edition: entry 7717). These adjuvant molecules are
described in WO 99/52549.
[0329] The polyoxyethylene ether according to the general formula
(I) above may, if desired, be combined with another adjuvant. For
example, a preferred adjuvant combination is preferably with CpG as
described in the pending UK patent application GB 9820956.2.
[0330] According to another embodiment of this invention, an
immunogenic composition described herein is delivered to a host via
antigen presenting cells (APCs), such as dendritic cells,
macrophages, B cells, monocytes and other cells that may be
engineered to be efficient APCs. Such cells may, but need not, be
genetically modified to increase the capacity for presenting the
antigen, to improve activation and/or maintenance of the T cell
response, to have anti-tumor effects per se and/or to be
immunologically compatible with the receiver (i.e., matched HLA
haplotype). APCs may generally be isolated from any of a variety of
biological fluids and organs, including tumor and peritumoral
tissues, and may be autologous, allogeneic, syngeneic or xenogeneic
cells.
[0331] Certain preferred embodiments of the present invention use
dendritic cells or progenitors thereof as antigen-presenting cells.
Dendritic cells are highly potent APCs (Banchereau and Steinman,
Nature 392:245-251, 1998) and have been shown to be effective as a
physiological adjuvant for eliciting prophylactic or therapeutic
antitumor immunity (see Timmerman and Levy, Ann. Rev. Med.
50:507-529, 1999). In general, dendritic cells may be identified
based on their typical shape (stellate in situ, with marked
cytoplasmic processes (dendrites) visible in vitro), their ability
to take up, process and present antigens with high efficiency and
their ability to activate nave T cell responses. Dendritic cells
may, of course, be engineered to express specific cell-surface
receptors or ligands that are not commonly found on dendritic cells
in vivo or ex vivo, and such modified dendritic cells are
contemplated by the present invention. As an alternative to
dendritic cells, secreted vesicles antigen-loaded dendritic cells
(called exosomes) may be used within a vaccine (see Zitvogel et
al., Nature Med. 4:594-600, 1998).
[0332] Dendritic cells and progenitors may be obtained from
peripheral blood, bone marrow, tumor-infiltrating cells,
peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin,
umbilical cord blood or any other suitable tissue or fluid. For
example, dendritic cells may be differentiated ex vivo by adding a
combination of cytokines such as GM-CSF, IL-4, IL-13 and/or
TNF.alpha. to cultures of monocytes harvested from peripheral
blood. Alternatively, CD34 positive cells harvested from peripheral
blood, umbilical cord blood or bone marrow may be differentiated
into dendritic cells by adding to the culture medium combinations
of GM-CSF, IL-3, TNF.alpha., CD40 ligand, LPS, flt3 ligand and/or
other compound(s) that induce differentiation, maturation and
proliferation of dendritic cells.
[0333] Dendritic cells are conveniently categorized as "immature"
and "mature" cells, which allows a simple way to discriminate
between two well characterized phenotypes. However, this
nomenclature should not be construed to exclude all possible
intermediate stages of differentiation. Immature dendritic cells
are characterized as APC with a high capacity for antigen uptake
and processing, which correlates with the high expression of
Fc.gamma. receptor and mannose receptor. The mature phenotype is
typically characterized by a lower expression of these markers, but
a high expression of cell surface molecules responsible for T cell
activation such as class I and class II MHC, adhesion molecules
(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40,
CD80, CD86 and 4-1BB).
[0334] APCs may generally be transfected with a polynucleotide of
the invention (or portion or other variant thereof) such that the
encoded polypeptide, or an immunogenic portion thereof, is
expressed on the cell surface. Such transfection may take place ex
vivo, and a pharmaceutical composition comprising such transfected
cells may then be used for therapeutic purposes, as described
herein. Alternatively, a gene delivery vehicle that targets a
dendritic or other antigen presenting cell may be administered to a
patient, resulting in transfection that occurs in vivo. In vivo and
ex vivo transfection of dendritic cells, for example, may generally
be performed using any methods known in the art, such as those
described in WO 97/24447, or the gene gun approach described by
Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen
loading of dendritic cells may be achieved by incubating dendritic
cells or progenitor cells with the tumor polypeptide, DNA (naked or
within a plasmid vector) or RNA; or with antigen-expressing
recombinant bacterium or viruses (e.g., vaccinia, fowlpox,
adenovirus or lentivirus vectors). Prior to loading, the
polypeptide may be covalently conjugated to an immunological
partner that provides T cell help (e.g., a carrier molecule).
Alternatively, a dendritic cell may be pulsed with a non-conjugated
immunological partner, separately or in the presence of the
polypeptide.
[0335] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will typically vary depending
on the mode of administration. Compositions of the present
invention may be formulated for any appropriate manner of
administration, including for example, topical, oral, nasal,
mucosal, intravenous, intracranial, intraperitoneal, subcutaneous
and intramuscular administration.
[0336] Carriers for use within such pharmaceutical compositions are
biocompatible, and may also be biodegradable. In certain
embodiments, the formulation preferably provides a relatively
constant level of active component release. In other embodiments,
however, a more rapid rate of release immediately upon
administration may be desired. The formulation of such compositions
is well within the level of ordinary skill in the art using known
techniques. Illustrative carriers useful in this regard include
microparticles of poly(lactide-co-glycolide), polyacrylate, latex,
starch, cellulose, dextran and the like. Other illustrative
delayed-release carriers include supramolecular biovectors, which
comprise a non-liquid hydrophilic core (e.g., a cross-linked
polysaccharide or oligosaccharide) and, optionally, an external
layer comprising an amphiphilic compound, such as a phospholipid
(see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO
94/20078, WO/94/23701 and WO 96/06638). The amount of active
compound contained within a sustained release formulation depends
upon the site of implantation, the rate and expected duration of
release and the nature of the condition to be treated or
prevented.
[0337] In another illustrative embodiment, biodegradable
microspheres (e.g., polylactate polyglycolate) are employed as
carriers for the compositions of this invention. Suitable
biodegradable microspheres are disclosed, for example, in U.S. Pat.
Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883;
5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B
core protein carrier systems. such as described in WO/99 40934, and
references cited therein, will also be useful for many
applications. Another illustrative carrier/delivery system employs
a carrier comprising particulate-protein complexes, such as those
described in U.S. Pat. No. 5,928,647, which are capable of inducing
a class I-restricted cytotoxic T lymphocyte responses in a
host.
[0338] In another illustrative embodiment, calcium phosphate core
particles are employed as carriers, vaccine adjuvants, or as
controlled release matrices for the compositions of this invention.
Exemplary calcium phosphate particles are disclosed, for example,
in published patent application No. WO/0046147.
[0339] The pharmaceutical compositions of the invention will often
further comprise one or more buffers (e.g., neutral buffered saline
or phosphate buffered saline), carbohydrates (e.g., glucose,
mannose, sucrose or dextrans), mannitol, proteins, polypeptides or
amino acids such as glycine, antioxidants, bacteriostats, chelating
agents such as EDTA or glutathione, adjuvants (e.g., aluminum
hydroxide), solutes that render the formulation isotonic, hypotonic
or weakly hypertonic with the blood of a recipient, suspending
agents, thickening agents and/or preservatives. Alternatively,
compositions of the present invention may be formulated as a
lyophilizate.
[0340] The pharmaceutical compositions described herein may be
presented in unit-dose or multi-dose containers, such as sealed
ampoules or vials. Such containers are typically sealed in such a
way to preserve the sterility and stability of the formulation
until use. In general, formulations may be stored as suspensions,
solutions or emulsions in oily or aqueous vehicles. Alternatively,
a pharmaceutical composition may be stored in a freeze-dried
condition requiring only the addition of a sterile liquid carrier
immediately prior to use.
[0341] The development of suitable dosing and treatment regimens
for using the particular compositions described herein in a variety
of treatment regimens, including e.g., oral, parenteral,
intravenous, intranasal, and intramuscular administration and
formulation, is well known in the art, some of which are briefly
discussed below for general purposes of illustration.
[0342] In certain applications, the pharmaceutical compositions
disclosed herein may be delivered via oral administration to an
animal. As such, these compositions may be formulated with an inert
diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0343] The active compounds may even be incorporated with
excipients and used in the form of ingestible tablets, buccal
tables, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like (see, for example, Mathiowitz et al., Nature 1997 Mar
27;386(6623):410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst
1998;15(3):243-84; U.S. Pat. No. 5,641,515; U.S. Pat. No. 5,580,579
and U.S. Pat. No. 5,792,451). Tablets, troches, pills, capsules and
the like may also contain any of a variety of additional
components, for example, a binder, such as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar, or both. Of course, any material used in preparing any
dosage unit form should be pharmaceutically pure and substantially
non-toxic in the amounts employed. In addition, the active
compounds may be incorporated into sustained-release preparation
and formulations.
[0344] Typically, these formulations will contain at least about
0.1% of the active compound or more, although the percentage of the
active ingredient(s) may, of course, be varied and may conveniently
be between about 1 or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. Naturally, the amount of active
compound(s) in each therapeutically useful composition may be
prepared is such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
[0345] For oral administration the compositions of the present
invention may alternatively be incorporated with one or more
excipients in the form of a mouthwash, dentifrice, buccal tablet,
oral spray, or sublingual orally-administered formulation.
Alternatively, the active ingredient may be incorporated into an
oral solution such as one containing sodium borate, glycerin and
potassium bicarbonate, or dispersed in a dentifrice, or added in a
therapeutically-effective amount to a composition that may include
water, binders, abrasives, flavoring agents, foaming agents, and
humectants. Alternatively the compositions may be fashioned into a
tablet or solution form that may be placed under the tongue or
otherwise dissolved in the mouth.
[0346] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or even intraperitoneally. Such
approaches are well known to the skilled artisan, some of which are
further described, for example, in U.S. Pat. No. 5,543,158; U.S.
Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain
embodiments, solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations generally will
contain a preservative to prevent the growth of microorganisms.
[0347] Illustrative pharmaceutical forms suitable for injectable
use include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions (for example, see U.S. Pat. No.
5,466,468). In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and/or vegetable oils. Proper
fluidity may be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and/or by the use of surfactants. The
prevention of the action of microorganisms can be facilitated by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0348] In one embodiment, for parenteral administration in an
aqueous solution, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this
connection, a sterile aqueous medium that can be employed will be
known to those of skill in the art in light of the present
disclosure. For example, one dosage may be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. Moreover, for human administration, preparations
will of course preferably meet sterility, pyrogenicity, and the
general safety and purity standards as required by FDA Office of
Biologics standards.
[0349] In another embodiment of the invention, the compositions
disclosed herein may be formulated in a neutral or salt form.
Illustrative pharmaceutically-acceptable salts include the acid
addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective.
[0350] The carriers can further comprise any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a human.
[0351] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering genes, nucleic acids, and
peptide compositions directly to the lungs via nasal aerosol sprays
has been described, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat.
No. 5,804,212. Likewise, the delivery of drugs using intranasal
microparticle resins (Takenaga et al., J Controlled Release 1998
Mar 2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S.
Pat. No. 5,725,871) are also well-known in the pharmaceutical arts.
Likewise, illustrative transmucosal drug delivery in the form of a
polytetrafluoroetheylene support matrix is described in U.S. Pat.
No. 5,780,045.
[0352] In certain embodiments, liposomes, nanocapsules,
microparticles, lipid particles, vesicles, and the like, are used
for the introduction of the compositions of the present invention
into suitable host cells/organisms. In particular, the compositions
of the present invention may be formulated for delivery either
encapsulated in a lipid particle, a liposome, a vesicle, a
nanosphere, or a nanoparticle or the like. Alternatively,
compositions of the present invention can be bound, either
covalently or non-covalently, to the surface of such carrier
vehicles.
[0353] The formation and use of liposome and liposome-like
preparations as potential drug carriers is generally known to those
of skill in the art (see for example, Lasic, Trends Biotechnol 1998
Jul;16(7):307-21; Takakura, Nippon Rinsho 1998 Mar;56(3):691-5;
Chandran et al., Indian J Exp Biol. 1997 Aug;35(8):801-9; Margalit,
Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Pat. No.
5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S.
Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each specifically
incorporated herein by reference in its entirety).
[0354] Liposomes have been used successfully with a number of cell
types that are normally difficult to transfect by other procedures,
including T cell suspensions, primary hepatocyte cultures and PC 12
cells (Renneisen et al., J Biol Chem. 1990 Sep 25;265(27):16337-42;
Muller et al., DNA Cell Biol. 1990 Apr;9(3):221-9). In addition,
liposomes are free of the DNA length constraints that are typical
of viral-based delivery systems. Liposomes have been used
effectively to introduce genes, various drugs, radiotherapeutic
agents, enzymes, viruses, transcription factors, allosteric
effectors and the like, into a variety of cultured cell lines and
animals. Furthermore, he use of liposomes does not appear to be
associated with autoimmune responses or unacceptable toxicity after
systemic delivery.
[0355] In certain embodiments, liposomes are formed from
phospholipids that are dispersed in an aqueous medium and
spontaneously form multilamellar concentric bilayer vesicles (also
termed multilamellar vesicles (MLVs).
[0356] Alternatively, in other embodiments, the invention provides
for pharmaceutically-acceptable nanocapsule formulations of the
compositions of the present invention. Nanocapsules can generally
entrap compounds in a stable and reproducible way (see, for
example, Quintanar-Guerrero et al., Drug Dev Ind Pharm. 1998
Dec;24(12):1113-28). To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) may be designed using polymers able to be degraded in vivo.
Such particles can be made as described, for example, by Couvreur
et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen
et al., Eur J Pharm Biopharm. 1998 Mar;45(2):149-55; Zambaux et al.
J Controlled Release. 1998 Jan 2;50(1-3):31-40; and U.S. Pat. No.
5,145,684.
[0357] Cancer Therapeutic Methods
[0358] Immunologic approaches to cancer therapy are based on the
recognition that cancer cells can often evade the body's defenses
against aberrant or foreign cells and molecules, and that these
defenses might be therapeutically stimulated to regain the lost
ground, e.g. pgs. 623-648 in Klein, Immunology (Wiley-Interscience,
New York, 1982). Numerous recent observations that various immune
effectors can directly or indirectly inhibit growth of tumors has
led to renewed interest in this approach to cancer therapy, e.g.
Jager, et al., Oncology 2001;60(1):1-7; Renner, et al., Ann Hematol
2000 Dec;79(12):651-9.
[0359] Four-basic cell types whose function has been associated
with antitumor cell immunity and the elimination of tumor cells
from the body are: i) B-lymphocytes which secrete immunoglobulins
into the blood plasma for identifying and labeling the nonself
invader cells; ii) monocytes which secrete the complement proteins
that are responsible for lysing and processing the
immunoglobulin-coated target invader cells; iii) natural killer
lymphocytes having two mechanisms for the destruction of tumor
cells, antibody-dependent cellular cytotoxicity and natural
killing; and iv) T-lymphocytes possessing antigen-specific
receptors and having the capacity to recognize a tumor cell
carrying complementary marker molecules (Schreiber, H., 1989, in
Fundamental Immunology (ed). W. E. Paul, pp. 923-955).
[0360] Cancer immunotherapy generally focuses on inducing humoral
immune responses, cellular immune responses, or both. Moreover, it
is well established that induction of CD4.sup.+ T helper cells is
necessary in order to secondarily induce either antibodies or
cytotoxic CD8.sup.+ T cells. Polypeptide antigens that are
selective or ideally specific for cancer cells, particularly lung
cancer cells, offer a powerful approach for inducing immune
responses against lung cancer, and are an important aspect of the
present invention.
[0361] Therefore, in further aspects of the present invention, the
pharmaceutical compositions described herein may be used to
stimulate an immune response against cancer, particularly for the
immunotherapy of lung cancer. Within such methods, the
pharmaceutical compositions described herein are administered to a
patient, typically a warm-blooded animal, preferably a human. A
patient may or may not be afflicted with cancer. Pharmaceutical
compositions and vaccines may be administered either prior to or
following surgical removal of primary tumors and/or treatment such
as administration of radiotherapy or conventional chemotherapeutic
drugs. As discussed above, administration of the pharmaceutical
compositions may be by any suitable method, including
administration by intravenous, intraperitoneal, intramuscular,
subcutaneous, intranasal, intradermal, anal, vaginal, topical and
oral routes.
[0362] Within certain embodiments, immunotherapy may be active
immunotherapy, in which treatment relies on the in vivo stimulation
of the endogenous host immune system to react against tumors with
the administration of immune response-modifying agents (such as
polypeptides and polynucleotides as provided herein).
[0363] Within other embodiments, immunotherapy may be passive
immunotherapy, in which treatment involves the delivery of agents
with established tumor-immune reactivity (such as effector cells or
antibodies) that can directly or indirectly mediate antitumor
effects and does not necessarily depend on an intact host immune
system. Examples of effector cells include T cells as discussed
above, T lymphocytes (such as CD8.sup.+ cytotoxic T lymphocytes and
CD4.sup.+ T-helper tumor-infiltrating lymphocytes), killer cells
(such as Natural Killer cells and lymphokine-activated killer
cells), B cells and antigen-presenting cells (such as dendritic
cells and macrophages) expressing a polypeptide provided herein. T
cell receptors and antibody receptors specific for the polypeptides
recited herein may be cloned, expressed and transferred into other
vectors or effector cells for adoptive immunotherapy. The
polypeptides provided herein may also be used to generate
antibodies or anti-idiotypic antibodies (as described above and in
U.S. Pat. No. 4,918,164) for passive immunotherapy.
[0364] Monoclonal antibodies may be labeled with any of a variety
of labels for desired selective usages in detection, diagnostic
assays or therapeutic applications (as described in U.S. Pat. Nos.
6,090,365; 6,015,542; 5,843,398; 5,595,721; and 4,708,930, hereby
incorporated by reference in their entirety as if each was
incorporated individually). In each case, the binding of the
labelled monoclonal antibody to the determinant site of the antigen
will signal detection or delivery of a particular therapeutic agent
to the antigenic determinant on the non-normal cell. A further
object of this invention is to provide the specific monoclonal
antibody suitably labelled for achieving such desired selective
usages thereof.
[0365] Effector cells may generally be obtained in sufficient
quantities for adoptive immunotherapy by growth in vitro, as
described herein. Culture conditions for expanding single
antigen-specific effector cells to several billion in number with
retention of antigen recognition in vivo are well known in the art.
Such in vitro culture conditions typically use intermittent
stimulation with antigen, often in the presence of cytokines (such
as IL-2) and non-dividing feeder cells. As noted above,
immunoreactive polypeptides as provided herein may be used to
rapidly expand antigen-specific T cell cultures in order to
generate a sufficient number of cells for immunotherapy. In
particular, antigen-presenting cells, such as dendritic,
macrophage, monocyte, fibroblast and/or B cells, may be pulsed with
immunoreactive polypeptides or transfected with one or more
polynucleotides using standard techniques well known in the art.
For example, antigen-presenting cells can be transfected with a
polynucleotide having a promoter appropriate for increasing
expression in a recombinant virus or other expression system.
Cultured effector cells for use in therapy must be able to grow and
distribute widely, and to survive long term in vivo. Studies have
shown that cultured effector cells can be induced to grow in vivo
and to survive long term in substantial numbers by repeated
stimulation with antigen supplemented with IL-2 (see, for example,
Cheever et al., Immunological Reviews 157:177, 1997).
[0366] Alternatively, a vector expressing a polypeptide recited
herein may be introduced into antigen presenting cells taken from a
patient and clonally propagated ex vivo for transplant back into
the same patient. Transfected cells may be reintroduced into the
patient using any means known in the art, preferably in sterile
form by intravenous, intracavitary, intraperitoneal or intratumor
administration.
[0367] Routes and frequency of administration of the therapeutic
compositions described herein, as well as dosage, will vary from
individual to individual, and may be readily established using
standard techniques. In general, the pharmaceutical compositions
and vaccines may be administered by injection (e.g.,
intracutaneous, intramuscular, intravenous or subcutaneous),
intranasally (e.g., by aspiration) or orally. Preferably, between 1
and 10 doses may be administered over a 52 week period. Preferably,
6 doses are administered, at intervals of 1 month, and booster
vaccinations may be given periodically thereafter. Alternate
protocols may be appropriate for individual patients. A suitable
dose is an amount of a compound that, when administered as
described above, is capable of promoting an anti-tumor immune
response, and is at least 10-50% above the basal (i.e., untreated)
level. Such response can be monitored by measuring the anti-tumor
antibodies in a patient or by vaccine-dependent generation of
cytolytic effector cells capable of killing the patient's tumor
cells in vitro. Such vaccines should also be capable of causing an
immune response that leads to an improved clinical outcome (e.g.,
more frequent remissions, complete or partial or longer
disease-free survival) in vaccinated patients as compared to
non-vaccinated patients. In general, for pharmaceutical
compositions and vaccines comprising one or more polypeptides, the
amount of each polypeptide present in a dose ranges from about 25
.mu.g to 5 mg per kg of host. Suitable dose sizes will vary with
the size of the patient, but will typically range from about 0.1 mL
to about 5 mL.
[0368] In general, an appropriate dosage and treatment regimen
provides the active compound(s) in an amount sufficient to provide
therapeutic and/or prophylactic benefit. Such a response can be
monitored by establishing an improved clinical outcome (e.g., more
frequent remissions, complete or partial, or longer disease-free
survival) in treated patients as compared to non-treated patients.
Increases in preexisting immune responses to a tumor protein
generally correlate with an improved clinical outcome. Such immune
responses may generally be evaluated using standard proliferation,
cytotoxicity or cytokine assays, which may be performed using
samples obtained from a patient before and after treatment.
[0369] Cancer Detection and Diagnostic Compositions Methods and
Kits
[0370] In general, a cancer may be detected in a patient based on
the presence of one or more lung tumor proteins and/or
polynucleotides encoding such proteins in a biological sample (for
example, blood, sera, sputum urine and/or tumor biopsies) obtained
from the patient. In other words, such proteins may be used as
markers to indicate the presence or absence of a cancer such as
lung cancer. In addition, such proteins may be useful for the
detection of other cancers. The binding agents provided herein
generally permit detection of the level of antigen that binds to
the agent in the biological sample.
[0371] Polynucleotide primers and probes may be used to detect the
level of mRNA encoding a tumor protein, which is also indicative of
the presence or absence of a cancer. In general, a tumor sequence
should be present at a level that is at least two-fold, preferably
three-fold, and more preferably five-fold or higher in tumor tissue
than in normal tissue of the same type from which the tumor arose.
Expression levels of a particular tumor sequence in tissue types
different from that in which the tumor arose are irrelevant in
certain diagnostic embodiments since the presence of tumor cells
can be confirmed by observation of predetermined differential
expression levels, e.g., 2-fold, 5-fold, etc, in tumor tissue to
expression levels in normal tissue of the same type.
[0372] Other differential expression patterns can be utilized
advantageously for diagnostic purposes. For example, in one aspect
of the invention, overexpression of a tumor sequence in tumor
tissue and normal tissue of the same type, but not in other normal
tissue types, e.g. PBMCs, can be exploited diagnostically. In this
case, the presence of metastatic tumor cells, for example in a
sample taken from the circulation or some other tissue site
different from that in which the tumor arose, can be identified
and/or confirmed by detecting expression of the tumor sequence in
the sample, for example using RT-PCR analysis. In many instances,
it will be desired to enrich for tumor cells in the sample of
interest, e.g., PBMCs, using cell capture or other like
techniques.
[0373] There are a variety of assay formats known to those of
ordinary skill in the art for using a binding agent to detect
polypeptide markers in a sample. See, e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988. In general, the presence or absence of a cancer in a patient
may be determined by (a) contacting a biological sample obtained
from a patient with a binding agent; (b) detecting in the sample a
level of polypeptide that binds to the binding agent; and (c)
comparing the level of polypeptide with a predetermined cut-off
value.
[0374] In a preferred embodiment, the assay involves the use of
binding agent immobilized on a solid support to bind to and remove
the polypeptide from the remainder of the sample. The bound
polypeptide may then be detected using a detection reagent that
contains a reporter group and specifically binds to the binding
agent/polypeptide complex. Such detection reagents may comprise,
for example, a binding agent that specifically binds to the
polypeptide or an antibody or other agent that specifically binds
to the binding agent, such as an anti-immunoglobulin, protein G,
protein A or a lectin. Alternatively, a competitive assay may be
utilized, in which a polypeptide is labeled with a reporter group
and allowed to bind to the immobilized binding agent after
incubation of the binding agent with the sample. The extent to
which components of the sample inhibit the binding of the labeled
polypeptide to the binding agent is indicative of the reactivity of
the sample with the immobilized binding agent. Suitable
polypeptides for use within such assays include full length lung
tumor proteins and polypeptide portions thereof to which the
binding agent binds, as described above.
[0375] The solid support may be any material known to those of
ordinary skill in the art to which the tumor protein may be
attached. For example, the solid support may be a test well in a
microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, the support may be a bead or disc, such as glass,
fiberglass, latex or a plastic material such as polystyrene or
polyvinylchloride. The support may also be a magnetic particle or a
fiber optic sensor, such as those disclosed, for example, in U.S.
Pat. No. 5,359,681. The binding agent may be immobilized on the
solid support using a variety of techniques known to those of skill
in the art, which are amply described in the patent and scientific
literature. In the context of the present invention, the term
"immobilization" refers to both noncovalent association, such as
adsorption, and covalent attachment (which may be a direct linkage
between the agent and functional groups on the support or may be a
linkage by way of a cross-linking agent). Immobilization by
adsorption to a well in a microtiter plate or to a membrane is
preferred. In such cases, adsorption may be achieved by contacting
the binding agent, in a suitable buffer, with the solid support for
a suitable amount of time. The contact time varies with
temperature, but is typically between about 1 hour and about 1 day.
In general, contacting a well of a plastic microtiter plate (such
as polystyrene or polyvinylchloride) with an amount of binding
agent ranging from about 10 ng to about 10 .mu.g, and preferably
about 100 ng to about 1 .mu.g, is sufficient to immobilize an
adequate amount of binding agent.
[0376] Covalent attachment of binding agent to a solid support may
generally be achieved by first reacting the support with a
bifunctional reagent that will react with both the support and a
functional group, such as a hydroxyl or amino group, on the binding
agent. For example, the binding agent may be covalently attached to
supports having an appropriate polymer coating using benzoquinone
or by condensation of an aldehyde group on the support with an
amine and an active hydrogen on the binding partner (see, e.g.,
Pierce Immunotechnology Catalog and Handbook, 1991, at
A12-A13).
[0377] In certain embodiments, the assay is a two-antibody sandwich
assay. This assay may be performed by first contacting an antibody
that has been immobilized on a solid support, commonly the well of
a microtiter plate, with the sample, such that polypeptides within
the sample are allowed to bind to the immobilized antibody. Unbound
sample is then removed from the immobilized polypeptide-antibody
complexes and a detection reagent (preferably a second antibody
capable of binding to a different site on the polypeptide)
containing a reporter group is added. The amount of detection
reagent that remains bound to the solid support is then determined
using a method appropriate for the specific reporter group.
[0378] More specifically, once the antibody is immobilized on the
support as described above, the remaining protein binding sites on
the support are typically blocked. Any suitable blocking agent
known to those of ordinary skill in the art, such as bovine serum
albumin or Tween 20.TM. (Sigma Chemical Co., St. Louis, Mo.). The
immobilized antibody is then incubated with the sample, and
polypeptide is allowed to bind to the antibody. The sample may be
diluted with a suitable diluent, such as phosphate-buffered saline
(PBS) prior to incubation. In general, an appropriate contact time
(i.e., incubation time) is a period of time that is sufficient to
detect the presence of polypeptide within a sample obtained from an
individual with lung least about 95% of that achieved at
equilibrium between bound and unbound polypeptide. Those of
ordinary skill in the art will recognize that the time necessary to
achieve equilibrium may be readily determined by assaying the level
of binding that occurs over a period of time. At room temperature,
an incubation time of about 30 minutes is generally sufficient.
[0379] Unbound sample may then be removed by washing the solid
support with an appropriate buffer, such as PBS containing 0.1%
Tween 20.TM.. The second antibody, which contains a reporter group,
may then be added to the solid support. Preferred reporter groups
include those groups recited above.
[0380] The detection reagent is then incubated with the immobilized
antibody-polypeptide complex for an amount of time sufficient to
detect the bound polypeptide. An appropriate amount of time may
generally be determined by assaying the level of binding that
occurs over a period of time. Unbound detection reagent is then
removed and bound detection reagent is detected using the reporter
group. The method employed for detecting the reporter group depends
upon the nature of the reporter group. For radioactive groups,
scintillation counting or autoradiographic methods are generally
appropriate. Spectroscopic methods may be used to detect dyes,
luminescent groups and fluorescent groups. Biotin may be detected
using avidin, coupled to a different reporter group (commonly a
radioactive or fluorescent group or an enzyme). Enzyme reporter
groups may generally be detected by the addition of substrate
(generally for a specific period of time), followed by
spectroscopic or other analysis of the reaction products.
[0381] To determine the presence or absence of a cancer, such as
lung cancer, the signal detected from the reporter group that
remains bound to the solid support is generally compared to a
signal that corresponds to a predetermined cut-off value. In one
preferred embodiment, the cut-off value for the detection of a
cancer is the average mean signal obtained when the immobilized
antibody is incubated with samples from patients without the
cancer. In general, a sample generating a signal that is three
standard deviations above the predetermined cut-off value is
considered positive for the cancer. In an alternate preferred
embodiment, the cut-off value is determined using a Receiver
Operator Curve, according to the method of Sackett et al., Clinical
Epidemiology: A Basic Science for Clinical Medicine, Little Brown
and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off
value may be determined from a plot of pairs of true positive rates
(i.e., sensitivity) and false positive rates (100%-specificity)
that correspond to each possible cut-off value for the diagnostic
test result. The cut-off value on the plot that is the closest to
the upper left-hand corner (i.e., the value that encloses the
largest area) is the most accurate cut-off value, and a sample
generating a signal that is higher than the cut-off value
determined by this method may be considered positive.
Alternatively, the cut-off value may be shifted to the left along
the plot, to minimize the false positive rate, or to the right, to
minimize the false negative rate. In general, a sample generating a
signal that is higher than the cut-off value determined by this
method is considered positive for a cancer.
[0382] In a related embodiment, the assay is performed in a
flow-through or strip test format, wherein the binding agent is
immobilized on a membrane, such as nitrocellulose. In the
flow-through test, polypeptides within the sample bind to the
immobilized binding agent as the sample passes through the
membrane. A second, labeled binding agent then binds to the binding
agent-polypeptide complex as a solution containing the second
binding agent flows through the membrane. The detection of bound
second binding agent may then be performed as described above. In
the strip test format, one end of the membrane to which binding
agent is bound is immersed in a solution containing the sample. The
sample migrates along the membrane through a region containing
second binding agent and to the area of immobilized binding agent.
Concentration of second binding agent at the area of immobilized
antibody indicates the presence of a cancer. Typically, the
concentration of second binding agent at that site generates a
pattern, such as a line, that can be read visually. The absence of
such a pattern indicates a negative result. In general, the amount
of binding agent immobilized on the membrane is selected to
generate a visually discernible pattern when the biological sample
contains a level of polypeptide that would be sufficient to
generate a positive signal in the two-antibody sandwich assay, in
the format discussed above. Preferred binding agents for use in
such assays are antibodies and antigen-binding fragments thereof.
Preferably, the amount of antibody immobilized on the membrane
ranges from about 25 ng to about 1 .mu.g, and more preferably from
about 50 ng to about 500 ng. Such tests can typically be performed
with a very small amount of biological sample.
[0383] Of course, numerous other assay protocols exist that are
suitable for use with the tumor proteins or binding agents of the
present invention. The above descriptions are intended to be
exemplary only. For example, it will be apparent to those of
ordinary skill in the art that the above protocols may be readily
modified to use tumor polypeptides to detect antibodies that bind
to such polypeptides in a biological sample. The detection of such
tumor protein specific antibodies may correlate with the presence
of a cancer.
[0384] A cancer may also, or alternatively, be detected based on
the presence of T cells that specifically react with a tumor
protein in a biological sample. Within certain methods, a
biological sample comprising CD4.sup.+ and/or CD8.sup.+ T cells
isolated from a patient is incubated with a tumor polypeptide, a
polynucleotide encoding such a polypeptide and/or an APC that
expresses at least an immunogenic portion of such a polypeptide,
and the presence or absence of specific activation of the T cells
is detected. Suitable biological samples include, but are not
limited to, isolated T cells. For example, T cells may be isolated
from a patient by routine techniques (such as by Ficoll/Hypaque
density gradient centrifugation of peripheral blood lymphocytes). T
cells may be incubated in vitro for 2-9 days (typically 4 days) at
37.degree. C. with polypeptide (e.g., 5-25 .mu.g/ml). It may be
desirable to incubate another aliquot of a T cell sample in the
absence of tumor polypeptide to serve as a control. For CD4.sup.+ T
cells, activation is preferably detected by evaluating
proliferation of the T cells. For CD8.sup.+ T cells, activation is
preferably detected by evaluating cytolytic activity. A level of
proliferation that is at least two fold greater and/or a level of
cytolytic activity that is at least 20% greater than in
disease-free patients indicates the presence of a cancer in the
patient.
[0385] As noted above, a cancer may also, or alternatively, be
detected based on the level of mRNA encoding a tumor protein in a
biological sample. For example, at least two oligonucleotide
primers may be employed in a polymerase chain reaction (PCR) based
assay to amplify a portion of a tumor cDNA derived from a
biological sample, wherein at least one of the oligonucleotide
primers is specific for (i.e., hybridizes to) a polynucleotide
encoding the tumor protein. The amplified cDNA is then separated
and detected using techniques well known in the art, such as gel
electrophoresis.
[0386] Similarly, oligonucleotide probes that specifically
hybridize to a polynucleotide encoding a tumor protein may be used
in a hybridization assay to detect the presence of polynucleotide
encoding the tumor protein in a biological sample.
[0387] To permit hybridization under assay conditions,
oligonucleotide primers and probes should comprise an
oligonucleotide sequence that has at least about 60%, preferably at
least about 75% and more preferably at least about 90%, identity to
a portion of a polynucleotide encoding a tumor protein of the
invention that is at least 10 nucleotides, and preferably at least
20 nucleotides, in length. Preferably, oligonucleotide primers
and/or probes hybridize to a polynucleotide encoding a polypeptide
described herein under moderately stringent conditions, as defined
above. Oligonucleotide primers and/or probes which may be usefully
employed in the diagnostic methods described herein preferably are
at least 10-40 nucleotides in length. In a preferred embodiment,
the oligonucleotide primers comprise at least 10 contiguous
nucleotides, more preferably at least 15 contiguous nucleotides, of
a DNA molecule having a sequence as disclosed herein. Techniques
for both PCR based assays and hybridization assays are well known
in the art (see, for example, Mullis et al., Cold Spring Harbor
Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology,
Stockton Press, NY, 1989).
[0388] One preferred assay employs RT-PCR, in which PCR is applied
in conjunction with reverse transcription. Typically, RNA is
extracted from a biological sample, such as biopsy tissue, and is
reverse transcribed to produce cDNA molecules. PCR amplification
using at least one specific primer generates a cDNA molecule, which
may be separated and visualized using, for example, gel
electrophoresis. Amplification may be performed on biological
samples taken from a test patient and from an individual who is not
afflicted with a cancer. The amplification reaction may be
performed on several dilutions of cDNA spanning two orders of
magnitude. A two-fold or greater increase in expression in several
dilutions of the test patient sample as compared to the same
dilutions of the non-cancerous sample is typically considered
positive.
[0389] In another aspect of the present invention, cell capture
technologies may be used in conjunction, with, for example,
real-time PCR to provide a more sensitive tool for detection of
metastatic cells expressing lung tumor antigens. Detection of lung
cancer cells in biological samples, e.g., bone marrow samples,
peripheral blood, and small needle aspiration samples is desirable
for diagnosis and prognosis in lung cancer patients.
[0390] Immunomagnetic beads coated with specific monoclonal
antibodies to surface cell markers, or tetrameric antibody
complexes, may be used to first enrich or positively select cancer
cells in a sample. Various commercially available kits may be used,
including Dynabeads.RTM. Epithelial Enrich (Dynal Biotech, Oslo,
Norway), StemSep.TM. (StemCell Technologies, Inc., Vancouver, BC),
and RosetteSep (StemCell Technologies). A skilled artisan will
recognize that other methodologies and kits may also be used to
enrich or positively select desired cell populations.
Dynabeads.RTM. Epithelial Enrich contains magnetic beads coated
with mAbs specific for two glycoprotein membrane antigens expressed
on normal and neoplastic epithelial tissues. The coated beads may
be added to a sample and the sample then applied to a magnet,
thereby capturing the cells bound to the beads. The unwanted cells
are washed away and the magnetically isolated cells eluted from the
beads and used in further analyses.
[0391] RosetteSep can be used to enrich cells directly from a blood
sample and consists of a cocktail of tetrameric antibodies that
targets a variety of unwanted cells and crosslinks them to
glycophorin A on red blood cells (RBC) present in the sample,
forming rosettes. When centrifuged over Ficoll, targeted cells
pellet along with the free RBC. The combination of antibodies in
the depletion cocktail determines which cells will be removed and
consequently which cells will be recovered. Antibodies that are
available include, but are not limited to: CD2, CD3, CD4, CD5, CD8,
CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25, CD29, CD33,
CD34, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66B, CD66e,
HLA-DR, IgE, and TCR.alpha..beta..
[0392] Additionally, it is contemplated in the present invention
that mAbs specific for lung tumor antigens can be generated and
used in a similar manner. For example, mAbs that bind to
tumor-specific cell surface antigens may be conjugated to magnetic
beads, or formulated in a tetrameric antibody complex, and used to
enrich or positively select metastatic lung tumor cells from a
sample. Once a sample is enriched or positively selected, cells may
be lysed and RNA isolated. RNA may then be subjected to RT-PCR
analysis using lung tumor-specific primers in a real-time PCR assay
as described herein. One skilled in the art will recognize that
enriched or selected populations of cells may be analyzed by other
methods (e.g. in situ hybridization or flow cytometry).
[0393] In another embodiment, the compositions described herein may
be used as markers for the progression of cancer. In this
embodiment, assays as described above for the diagnosis of a cancer
may be performed over time, and the change in the level of reactive
polypeptide(s) or polynucleotide(s) evaluated. For example, the
assays may be performed every 24-72 hours for a period of 6 months
to 1 year, and thereafter performed as needed. In general, a cancer
is progressing in those patients in whom the level of polypeptide
or polynucleotide detected increases over time. In contrast, the
cancer is not progressing when the level of reactive polypeptide or
polynucleotide either remains constant or decreases with time.
[0394] Certain in vivo diagnostic assays may be performed directly
on a tumor. One such assay involves contacting tumor cells with a
binding agent. The bound binding agent may then be detected
directly or indirectly via a reporter group. Such binding agents
may also be used in histological applications. Alternatively,
polynucleotide probes may be used within such applications.
[0395] As noted above, to improve sensitivity, multiple tumor
protein markers may be assayed within a given sample. It will be
apparent that binding agents specific for different proteins
provided herein may be combined within a single assay. Further,
multiple primers or probes may be used concurrently. The selection
of tumor protein markers may be based on routine experiments to
determine combinations that results in optimal sensitivity. In
addition, or alternatively, assays for tumor proteins provided
herein may be combined with assays for other known tumor
antigens.
[0396] The present invention further provides kits for use within
any of the above diagnostic methods. Such kits typically comprise
two or more components necessary for performing a diagnostic assay.
Components may be compounds, reagents, containers and/or equipment.
For example, one container within a kit may contain a monoclonal
antibody or fragment thereof that specifically binds to a tumor
protein. Such antibodies or fragments may be provided attached to a
support material, as described above. One or more additional
containers may enclose elements, such as reagents or buffers, to be
used in the assay. Such kits may also, or alternatively, contain a
detection reagent as described above that contains a reporter group
suitable for direct or indirect detection of antibody binding.
[0397] Alternatively, a kit may be designed to detect the level of
mRNA encoding a tumor protein in a biological sample. Such kits
generally comprise at least one oligonucleotide probe or primer, as
described above, that hybridizes to a polynucleotide encoding a
tumor protein. Such an oligonucleotide may be used, for example,
within a PCR or hybridization assay. Additional components that may
be present within such kits include a second oligonucleotide and/or
a diagnostic reagent or container to facilitate the detection of a
polynucleotide encoding a tumor protein.
[0398] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Identification of Lung Tumor Protein cDNAs
[0399] Lung-specific genes were identified by electronic
subtraction. The method used was similar to that described by
Vasmatizis et al., Proc. Natl. Acad. Sci. USA 95:300-304, 1998, but
there were several key differences. Sequences of EST clones
(1,453,679) were downloaded from the GenBank public human EST
database. Human cDNA libraries were downloaded to create a database
of these cDNA libraries and the EST sequences derived from them.
The cDNA libraries were grouped into three groups: Plus, Minus and
Other/Neutral. The Plus group included 30 libraries constructed
from lung tumor and fetal lung tissues (and therefore including
those containing lung tumor-specific ESTs); the Minus group
consisted of 206 libraries derived from all adult normal tissues;
the Other/Neutral group contained libraries from tissues where
expression is considered irrelevant (e.g., non-lung-fetal tissue,
non-lung tumors, cell lines other than lung tumor cell lines). A
total of 93,526 ESTs were derived from the 30 lung tumor and fetal
lung libraries. These ESTs were preprocessed to remove common
sequence repeats and cloning adapters, resulting in a final Plus
group of 90,365 (a decrease of 3%).
[0400] Each Plus group (lung tumor or fetal lung) EST sequence was
used as a query "seed" sequence in a BLASTN (version 2.0.9; May 7,
1999) search against the total human EST database. Standard
measures of similarity are insufficient in this sort of analysis,
as EST relationships often include short stretches and poor
sequence data. Criteria employed in this study required a matching
segment to be at least 75 nucleotides in length, and the density of
exact matches within this segment to be at least 80%. This was
considered conservative criteria designed to avoid short spurious
matches while allowing for polymorphisms and errors in sequencing.
Each BLAST search generated a cluster of related sequences based on
direct overlap with the query "seed" sequence. A second level of
clustering was performed to merge closely related clusters and to
eliminate redundancy resulting from the fact that similar clusters
are generated if the clusters contain more than one seed (i.e.,
sequences from the Plus EST group). The resulting "super clusters"
were discarded if they grew in size to 200 or more ESTs, since
these probably represented repetitive elements that were not
removed by the initial preprocessing of the seeds, or highly
expressed genes such as those for ribosomal proteins. Superclusters
were merged if they shared at least one third of their
sequences.
[0401] The BLAST searches gave rise to a total of 49,154 clusters.
In the first super clustering stage, 18,665 clusters grew beyond
the limit of 200 clones. The remainder was reduced to a total of
30,489 super clusters. This number was reduced to 29,501 after
adjacent clusters were merged. Resulting super clusters were
analyzed to determine the tissue source of each EST clone contained
within it and this expression profile was used to classify the
superclusters into four groups: Type 1- this supercluster contains
EST clones found in the Plus group only, with no expression in the
Minus or Other/Neutral group libraries; Type 2--EST clones in the
supercluster are found in the Plus and Other/Neutral group
libraries, with no expression in the Minus group; Type 3--super
cluster EST clones found in all groups, but the number of ESTs in
the Plus group is higher than in either of the Minus or
Other/Neutral groups; Type 4--super cluster EST clones found in all
groups, but the number in the Plus group is higher than in the
Minus group with expression in the Other/Neutral group non
relevant. Sequences derived from the Plus library group that were
placed in Types 1, 2 and 3 superclusters resulted in 20,487
polynucleotide sequences. The electronic subtraction procedures
identified these sequences as having significant differential
expression in lung tissue.
Example 2
Analysis of CDNA Expression Using Microarray Technology
[0402] 2208 of the clones identified from the lung electronic
subtraction procedure were evaluated for overexpression in specific
tumor tissues by microarray analysis. Using this approach, cDNA
sequences are PCR amplified and their mRNA expression profiles in
tumor and normal tissues are examined using cDNA microarray
technology essentially as described (Shena, M. et al., 1995 Science
270:467-70). In brief, the 2208 clones were arrayed onto glass
slides as multiple replicas, with each location corresponding to a
unique cDNA clone (as many as 5500 clones can be arrayed on a
single slide or chip). Each chip was hybridized with a pair of cDNA
probes that were fluorescence-labeled with Cy3 and Cy5,
respectively. Typically, 1 .mu.g of polyA.sup.+ RNA was used to
generate each cDNA probe. Since one cDNA probe is generated from
tumor tissue RNA and the other is generated from normal tissue RNA,
sequences that are differentially overexpressed in tumor tissue
will generate a stronger signal from the tumor specific probe than
the normal tissue probe, thus allowing the identification of those
sequences that exhibit elevated expression in tumor versus normal
tissue.
[0403] After hybridization, the chips were scanned and the
fluorescence intensity recorded for both Cy3 and Cy5 channels.
There were multiple built-in quality control steps. First, the
probe quality was monitored using a panel of 18 ubiquitously
expressed genes. Secondly, the control plate also had yeast DNA
fragments of which complementary RNA was spiked into the probe
synthesis for measuring the quality of the probe and the
sensitivity of the analysis. Currently, the technology offers a
sensitivity of 1 in 100,000 copies of mRNA. Finally, the
reproducibility of this technology was ensured by including
duplicated control cDNA elements at different locations. Further
validation of the process was indicated in that several
differentially expressed genes were identified multiple times in
the study, and the expression profiles for these genes are very
comparable. The clones were arrayed on Lung Chip 6.
[0404] Of those analyzed by microarray, 781 sequences met the
criteria of having at least 2-fold overexpression in lung tumor
tissue compared to normal tissues. Of these 781 clones, 459 were
found to meet the additional criteria of having a mean normal
tissue expression value less than or equal to 0.2. These 459 clones
were then analyzed visually and certain ones with favorable
expression profiles (e.g., high expression in tumors with little or
no expression in normal tissues) were sequenced and searched
against public sequences databases to facilitate identification of
extended sequence for the clones.
[0405] SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
29, 31, 32 and 34 represent a subset of those 459 clones that met
the above criteria of being at least 2-fold overexpressed in tumor
versus normal tissues and having a mean normal tissue expression of
less than or equal to 0.2. Additional information about these
sequences is provided in Table 2 below.
2TABLE 2 MICRO- MICRO- SEQ ID ARRAY ARRAY RATIO SEQ NO: ANALYSIS
(Lung ID from Clone Clone (Lung Tumor:Normal NO: 60/207,485 Name:
ID # Chip #) Tissue) 9 4538 L1027C 55571 6 2.94 5 4978 L1037C 58267
6 2.61 7 1796 L1038C 58245 6 3.5 3 7264 L1039C 58269 6 2.81 1 2337
L1040C 55964 6 5.07 15 1548/4619 L1041C 58346 6 2.33 25 15127 n/a
56016 6 >2 27 3816 n/a 55987 6 >2 29 2046 n/a 55956 6 >2
31 1912 n/a 55952 6 >2 32 2064 n/a 55957 6 >2 34 1502/3852
n/a 55559 6 >2 11 2814 n/a 55978 6 >2 13 3478 n/a 55980 6
>2 17 553 n/a 55561 6 >2 19 3275 n/a 55984 6 >2 21 2809
n/a 58261 6 >2 23 1677 n/a 58348 6 >2
[0406] Each of the sequences was then used as a query to search the
public databases in order to facilitate identification of extended
sequences for these clones. Extended sequence information for the
above sequences, obtained by searching public sequence databases,
is set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 33, and 35, respectively.
Example 3
Quantitative Real-time RT-PCR Analysis
[0407] Briefly, quantitation of PCR product relies on the few
cycles where the amount of DNA amplifies logarithmically from
barely above the background to the plateau. Using continuous
fluorescence monitoring, the threshold cycle number where DNA
amplifies logarithmically is easily determined in each PCR
reaction. There are two fluorescence detecting systems. One is
based upon a double-strand DNA specific binding dye SYBR Green I
dye. The other uses TaqMan probe containing a Reporter dye at the
5' end (FAM) and a Quencher dye at the 3' end (TAMRA) (Perkin
Elmer/Applied Biosystems Division, Foster City, Calif.).
Target-specific PCR amplification results in cleavage and release
of the Reporter dye from the Quencher-containing probe by the
nuclease activity of AmpliTaq Gold.TM. (Perkin Elmer/Applied
Biosystems Division, Foster City, Calif.). Thus, fluorescence
signal generated from released reporter dye is proportional to the
amount of PCR product. Both detection methods have been found to
generate comparable results. To compare the relative level of gene
expression in multiple tissue samples, a panel of cDNAs is
constructed using RNA from tissues and/or cell lines, and Real-Time
PCR is performed using gene specific primers to quantify the copy
number in each cDNA sample. Each cDNA sample is generally performed
in duplicate and each reaction repeated in duplicated plates. The
final Real-time PCR result is typically reported as an average of
copy number of a gene of interest normalized against internal actin
number in each cDNA sample. Real-time PCR reactions may be
performed on a GeneAmp 5700 Detector using SYBR Green I dye or an
ABI PRISM 7700 Detector using the TaqMan probe (Perkin
Elmer/Applied Biosystems Division, Foster City, Calif.).
[0408] Using this approach, Real Time PCRE profiles were generated
for L1027, L1037, L1038, L1039, L1040 and L1041, and are provided
in Table 3.
3TABLE 3 SEQ ID CLONE NO: NAME REAL TIME PROFILE 9 L1027C Real Time
PCR shows over-expression in small cell lung carcinoma as well as
in bone marrow. Expression is also observed for multiple normal
tissue. 5 L1037C Real Time PCR shows over-expression in small cell
lung carcinoma as well as in bone marrow and lymph node. Expression
is also observed for multiple normal tissue. 7 L1038C Real Time PCR
shows over-expression in small cell lung carcinoma as well as in
brain, pituitary gland and adrenal gland. Expression is also
observed for multiple normal tissue. 3 L1039C Real Time PCR shows
over-expression in small cell lung carcinoma as well as in lymph
node. Expression is also observed for multiple normal tissue. 1
L1040C Real Time PCR shows over-expression in small cell lung
carcinoma as well as in brain, pituitary gland and adrenal gland.
Expression is also observed for multiple normal tissue. 15 L0141C
Real Time PCR shows over-expression in small cell lung carcinoma as
well as in adrenal gland, bone marrow and thymus. Expression is
also observed for multiple normal tissue.
Example 4
Cloning of Full-length cDNA Sequences and ORF for L1027C
[0409] cDNA sequences encoding the full-length sequence for L1027C
were isolated by screening a small cell primary tumor full length
cloning library with a radioactively labeled probe of the original
isolate sequence (SEQ ID NO:9). In order to determine the
transcript size of the gene, a multiple tissue Northern blot was
probed with the radioactively labelled original isolate sequence,
SEQ ID NO:9. The Northern blot included 1 .mu.g of small cell
primary tumor polyA+ RNA. Visual analysis of the exposed film
revealed a single transcript of approximately 2.5 kb. Approximately
500,000 clones from the full-length cloning library were screened
and four clones were obtained from this library. The inserts were
sequenced and yielded DNA nucleotide molecules of about 2.32 and
2.37 kb. These sequences are provided in SEQ ID NO:93 and 94,
respectively. Both of these sequences contain the same single OFR
of 450 bp (SEQ ID NO:95), and encode a deduced amino acid sequence
of 150 amino acid residues (SEQ ID NO:96). These sequences were
searched against the Genbank nonredundant and GeneSeq DNA databases
and showed no hits.
Example 5
Analysis of cDNA Expression Using Microarray Technology
[0410] An additional 5054 of the resulting clones obtained from the
lung electronic subtraction of Example 1 were probed by microarray
chip technology to further characterize the expression of these
clones. The microarray analysis was carried out as provided in
Example 2. The clones were arrayed on Lung Chip 7. CorixArray
analysis was performed on the microarray results to compare
expression in lung tumors and in normal tissues. Clones were
selected based on two criteria: 2-fold overexpression in lung
tumors when compared to non-lung tissue and a mean expression level
of less than 0.2 in these same non-lung tissues. Of those analyzed,
2372 clones met the criteria.
[0411] Microarray analysis for five of these clones is presented in
Table 4:
4TABLE 4 MICRO- MICRO- SEQ ID ARRAY ARRAY RATIO SEQ NO: ANALYSIS
(Lung ID from Clone Clone (Lung Tumor:Normal NO: 60/207,485 Name:
ID # Chip #) Tissue) 42 18618 L1053C 63575 7 13.5 43 14788 L1054C
63582 7 5.29 44 7744 L1055C 63598 7 15.25 45 4257 L1056C 64963 7
9.31 46 20087 L1058C 64988 7 5.66
Example 6
Quantitative Real-time PCR Analysis
[0412] 170 of the 2372 clones of Example 4 were further analyzed by
visual analysis based on high expression in tumors and little or no
expression in normal tissues. Seven clones were selected for
Real-time PCR analysis. The Real-time PCR was carried out as
disclosed in Example 3. The Real-time PCR profiles of these seven
clones are presented in Table 5. The sequences of these seven
clones are provided in SEQ ID NO:42-48, respectively.
5TABLE 5 SEQ ID CLONE CLONE NO: NAME ID # REAL TIME PROFILE 42
L1053C 63575 Real Time PCR shows over-expression in small cell lung
carcinoma as well as in pituitary. Expression is also observed for
multiple normal tissues. 43 L1054C 63582 Real Time PCR shows
over-expression in small cell lung carcinoma as well as in
pituitary, brain and spinal cord. Expression is also observed for
adrenal and pancreas. 44 L1055C 63598 Real Time PCR shows
over-expression in small cell lung carcinoma as well as in
pituitary and brain. Expression is also observed for multiple
normal tissues. 45 L1056C 64963 Real Time PCR shows over-expression
in one small cell lung carcinoma sample. No expression is otherwise
observed. 46 L1058C 64988 Real Time PCR shows over-expression in
small cell lung carcinoma. Low level expression is also observed
for adrenal gland, pancreas, and bone marrow. 47 n/a 63485 Real
Time PCR shows over-expression in metastatic tumor as well as low
level expression in multiple normal tissues. 48 n/a 65010 Real Time
PCR shows low expression in one lung sample. No expression is
otherwise observed.
[0413] Each of the sequences was then used as a query to search the
public databases in order to facilitate identification of extended
sequences for these clones. SEQ ID NO:42, 43 and 45 matched to
known genes in Genbank, and these results are presented in Table 6.
The full-length cDNA sequences of the known genes are disclosed in
SEQ ID NO:49, 50 and 52, respectively. The deduced amino acid
sequences encoded by SEQ ID NO:49 and 50 are also provided as SEQ
ID NO:56 and 57, respectively. SEQ ID NO:44 and 46-48 were found to
be novel with respect to known genes, but matched to public EST
sequences. The sequences of SEQ ID NO:44 and 46-48 were aligned
with the matching EST sequences in order to obtain extended
sequence data. These extended sequences are provided in SEQ ID
NO:51 and 53-55, respectively.
6 TABLE 6 SEQ ID NO: CLONE NAME GENBANK DESCRIPTION 42 L1053C
Insulinoma-associated 1 43 L1054C KIAA0535 45 L1056C Human DAZ mRNA
3' UTR
Example 7
Cloning of cDNA Encoding Full-length L 1058C
[0414] The cDNA sequence encoding full-length L1058C was isolated
by screening a small cell primary tumor full length cloning library
with a radioactively labeled probe of the original isolate sequence
(SEQ ID NO:46). In order to determine the transcript size of the
gene, a multiple tissue Northern blot was probed with the
radioactively labelled original isolate sequence, SEQ ID NO:46. The
Northern blot included 1 .mu.g of small cell primary tumor,
carcinoid metastasis and small cell (tumor) cell line polyA+ RNA.
Visual analysis of the exposed film revealed a single transcript of
approximately 2.5 kb. Approximately 500,000 clones from the
full-length cloning library were screened and one clone was
obtained from this library. The insert was sequenced and yields a
2165 bp DNA nucleotide molecule. The full-length sequence is
provided in SEQ ID NO:58. The full-length sequence is predicted to
have two ORFs. A first ORF (SEQ ID NO:59) is predicted to encode a
polypeptide having 392 amino acid residues (SEQ ID NO:61), and the
second ORF (SEQ ID NO:60) is predicted to encode a polypeptide of
363 amino acid residues (SEQ ID NO:62) but does not show the
starting methionine. This 2165 bp DNA was searched against the
Genbank nonredundant and GeneSeq DNA databases and showed no
hits.
Example 8
Analysis of cDNA Expression Using Microarray Technology
[0415] An additional 3453 of the resulting clones obtained from the
lung electronic subtraction of Example 1 were probed by microarray
chip technology to further characterize the expression of these
clones. The microarray analysis was carried out as provided in
Example 2. The clones were arrayed on Lung Chip 8. CorixArray
analysis was performed on the microarray results to compare
expression in lung tumors and in normal tissues. Clones were
selected based on two criteria: 2-fold overexpression in lung
tumors when compared to non-lung tissue and a mean expression level
of less than 0.2 in these same non-lung tissues. Of those analyzed,
557 clones met the criteria.
[0416] 300 of the 557 clones were visually analyzed for
overexpression in tumor versus normal tissue. Twenty-eight clones
showing overexpression in tumor versus normal tissue were then
sequenced. These DNA sequences are provided in SEQ ID NO:63-92,
respectively. The microarray analysis for these 28 clones is
presented in Table 7.
7TABLE 7 MEDIAN MEDIAN SEQ ID NO: CLONE ID # RATIO SIGNAL 1 SIGNAL
2 63 72761 2.22 0.154 0.07 64 72762 2.33 0.105 0.045 65 72763 2.41
0.233 0.097 66 72764 2.72 0.199 0.073 67 72765 2.62 0.158 0.06 68
72766 2.84 0.149 0.053 69 72772 2.25 0.109 0.049 70 72775 2.36
0.103 0.044 71 72776 2.34 0.146 0.062 72 72779 2.25 0.22 0.098 73
72781 2.51 0.149 0.059 74 72784 2.35 0.212 0.09 75 72788 2.85 0.152
0.053 76 72789 2.69 0.196 0.073 77 72790 2.46 0.181 0.074 78 72791
2.39 0.143 0.06 79 72792 2.43 0.197 0.081 80 72794 3.04 0.258 0.085
81 72795 2.37 0.143 0.06 82 72797 2.96 0.233 0.079 83 72798 2.82
0.218 0.077 84 72804 2.33 0.14 0.06 85 72805 2.33 0.102 0.043 86
72806 2.32 0.121 0.052 87 72807 3.02 0.117 0.039 88 72808 2.74
0.109 0.04 89 72809 2.26 0.126 0.056 90 72811 2.92 0.151 0.052 91
72813 2.66 0.138 0.052 (L1080C)
[0417] Each of the sequences was then used as a query to search the
public sequence databases to identify novel and known genes.
Results of this search are provided in Table 8.
8TABLE 8 SEQ ID GEN BANK NO: ACC # GENESEQ DESCRIPTION 63 AC004590
Chromosome 17 64 Z78409 T62661 transcription factor E2F5 65 S45828
Z86797; cDNA DKFZp564L2416; A09328 nekl = serine/threonine-and
tyrosine-specific protein kinase [mice, erythroleukemia cells] 66
Novel 67 AL136169 Chromosome Xq26.1-27.1 68 AC011742 Chromosome 2,
AK021426 Homo sapiens cDNA FLJ11364 fis. clone HEMBA 1000264. 69 NM
005414 Q03742 SKI-like (SKIL) 70 NM 002335 V85551 low density
lipoprotein receptor- related protein 5 71 XM_004587 Homo sapiens
adaptor protein with pleckstrin homology and src homology 2 domains
(APS), AB000520 mRNA. Homo sapiens mRNA for APS, complete cds. 72
AK024119 cDNA FLJ14057 fis, clone HEMBB 1000337. 73 U86338 Mus
musculus zinc finger protein Png-1 (Png-1) 74 Novel 75 Novel 76
NM_002271 C03734 Homo sapiens karyopherin (importin) beta 3 (KPNB3)
mRNA 77 NM_001401 T48669; Homo sapiens endothelial T44104
differentiation, lysophosphatidic acid G-protein-coupled receptor,
2(EDG2), mRNA. 78 U40583 Human alpha/neuronal nicontinic
acetylcholine receptor mRNA, complete cds. 79 Z15509 Novel 80
Z59860 V34162 H. sapiens CpG island DNA genomic Msel fragment,
clone 178c7, reverse read cpg178c7.rtla. 81 Novel 82 Z59860 HNGIT2
DNA genomic Msel fragment, 2 clone 178c7 83 XM-004477 Q72451 Homo
sapiens glutamate-cysteine ligase, catalytic subunit (GCLC), mRNA.
84 Z16421 Novel 85 Novel 86 AC022013 V52850 Chromosome 3 87 Novel
88 AL354993 Z91766 Chromosome 20q13.2-13. Continas a peptidylprolyl
isomerase A (cyclophilin A) pseudogene, the gene for OVC10-2, ESTs,
STSs and GSSs, complete sequence 89 AC005021 Chromosome 7q21-q22,
complete sequence. 90 AK023904 cDNA FLJ13842 fis, clone
THYRO1000793.
Example 9
Quantitative Real-time PCR Analysis
[0418] One of the clones of Example 7, clone L1080C, was further
selected for Real-time PCR analysis. The Real-time PCR was carried
out as disclosed in Example 3. The Real-time PCR shows
over-expression in small cell lung carcinoma as well as in brain
and pituitary. Expression was also observed in thyroid, adrenal and
salivary glands.
Example 10
Identifying Full-length cDNA Sequence Encoding L1080C
[0419] The cDNA sequence encoding full-length L1080C was predicted
by using a partial sequence as a query to search the public
sequence databases to obtain extended sequence. The query resulted
in the identification of a full-length cDNA sequence for L1080C
(SEQ ID NO:91). The deduced amino acid sequence encoded by the
full-length cDNA sequence is provided in SEQ ID NO:92.
Example 11
Peptide Priming of T-helper Lines
[0420] Generation of CD4.sup.+ T helper lines and identification of
peptide epitopes derived from tumor-specific antigens that are
capable of being recognized by CD4.sup.+ T cells in the context of
HLA class II molecules, is carried out as follows:
[0421] Fifteen-mer peptides overlapping by 10 amino acids, derived
from a tumor-specific antigen, are generated using standard
procedures. Dendritic cells (DC) are derived from PBMC of a normal
donor using GM-CSF and IL-4 by standard protocols. CD4.sup.+ T
cells are generated from the same donor as the DC using MACS beads
(Miltenyi Biotec, Auburn, Calif.) and negative selection. DC are
pulsed overnight with pools of the 15-mer peptides, with each
peptide at a final concentration of 0.25 .mu.g/ml. Pulsed DC are
washed and plated at 1.times.10.sup.4 cells/well of 96-well
V-bottom plates and purified CD4.sup.+ T cells are added at
1.times.10.sup.5/well. Cultures are supplemented with 60 ng/ml IL-6
and 10 ng/ml IL-12 and incubated at 37.degree. C. Cultures are
restimulated as above on a weekly basis using DC generated and
pulsed as above as antigen presenting cells, supplemented with 5
ng/ml IL-7 and 10 U/ml IL-2. Following 4 in vitro stimulation
cycles, resulting CD4+T cell lines (each line corresponding to one
well) are tested for specific proliferation and cytokine production
in response to the stimulating pools of peptide with an irrelevant
pool of peptides used as a control.
Example 12
Generation of Tumor-specific CTL Lines Using in Vitro Whole-gene
Priming
[0422] Using in vitro whole-gene priming with tumor
antigen-vaccinia infected DC (see, for example, Yee et al, The
Journal of Immunology, 157(9):4079-86, 1996), human CTL lines are
derived that specifically recognize autologous fibroblasts
transduced with a specific tumor antigen, as determined by
interferon-.gamma. ELISPOT analysis. Specifically, dendritic cells
(DC) are differentiated from monocyte cultures derived from PBMC of
normal human donors by growing for five days in RPMI medium
containing 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml
human IL-4. Following culture, DC are infected overnight with tumor
antigen-recombinant vaccinia virus at a multiplicity of infection
(M.O.I) of five, and matured overnight by the addition of 3
.mu.g/ml CD40 ligand. Virus is then inactivated by UV irradiation.
CD8.sup.+ T cells are isolated using a magnetic bead system, and
priming cultures are initiated using standard culture techniques.
Cultures are restimulated every 7-10 days using autologous primary
fibroblasts retrovirally transduced with previously identified
tumor antigens. Following four stimulation cycles, CD8.sup.+ T cell
lines are identified that specifically produce interferon-.gamma.
when stimulated with tumor antigen-transduced autologous
fibroblasts. Using a panel of HLA-mismatched B-LCL lines transduced
with a vector expressing a tumor antigen, and measuring
interferon-.gamma. production by the CTL lines in an ELISPOT assay,
the HLA restriction of the CTL lines is determined.
Example 13
Generation and Characterization of Anti-tumor Antigen Monoclonal
Antibodies
[0423] Mouse monoclonal antibodies are raised against E. coli
derived tumor antigen proteins as follows: Mice are immunized with
Complete Freund's Adjuvant (CFA) containing 50 .mu.g recombinant
tumor protein, followed by a subsequent intraperitoneal boost with
Incomplete Freund's Adjuvant (IFA) containing 10 .mu.g recombinant
protein. Three days prior to removal of the spleens, the mice are
immunized intravenously with approximately 50 .mu.g of soluble
recombinant protein. The spleen of a mouse with a positive titer to
the tumor antigen is removed, and a single-cell suspension made and
used for fusion to SP2/O myeloma cells to generate B cell
hybridomas. The supernatants from the hybrid clones are tested by
ELISA for specificity to recombinant tumor protein, and epitope
mapped using peptides that span the entire tumor protein sequence.
The mAbs are also tested by flow cytometry for their ability to
detect tumor protein on the surface of cells stably transfected
with the cDNA encoding the tumor protein.
Example 14
Synthesis of Polypeptides
[0424] Polypeptides are synthesized on a Perkin Elmer/Applied
Biosystems Division 430A peptide synthesizer using FMOC chemistry
with HPTU (O-Benzotriazole-N,N,N',N'-tetramethyluronium
hexafluorophosphate) activation. A Gly-Cys-Gly sequence is attached
to the amino terminus of the peptide to provide a method of
conjugation, binding to an immobilized surface, or labeling of the
peptide. Cleavage of the peptides from the solid support is carried
out using the following cleavage mixture: trifluoroacetic
acid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After
cleaving for 2 hours, the peptides are precipitated in cold
methyl-t-butyl-ether. The peptide pellets are then dissolved in
water containing 0.1% trifluoroacetic acid (TFA) and lyophilized
prior to purification by C18 reverse phase HPLC. A gradient of
0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1%
TFA) is used to elute the peptides. Following lyophilization of the
pure fractions, the peptides are characterized using electrospray
or other types of mass spectrometry and by amino acid analysis.
[0425] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
96 1 644 DNA Homo sapiens misc_feature (1)...(644) n = A,T,C or G 1
ttactcctct agagggaaag catgacaccg aacactaagc acacagcttt ttgttgtttt
60 ggttttttct cccgcaaatc ttaaagtgat tcccatgacc ttggccaagg
acacttctta 120 aagattaatg actggcactg acattgcccc aggcgggcca
ctcctcacac tggctctcag 180 ttcccagcca tgcctggggc tcagtcactt
ctattccacc ctctgagact ccattggtgt 240 cacacaaggt gtcttcttgg
ctttgatttt gagaatcccc tattttcact tccagatctg 300 tcagctgcca
tggaggaata atagaaaacc agaaatgcgt gtagagggag atttctaaaa 360
cttcccttgt gtcgccatag ttgtagtttt gggttctggc aggtggaaca ccctgaaacc
420 tggaatcatt ctatgagaat acagttcaga ctttgcagac tccagcccat
actaactgtc 480 atgaagcttg acttcttgtc ataatgcagc catcttggag
gaaattggca tttctgctta 540 gatggntggc agggtcgcgc tcagctttgc
tttctacact aaattacata gcattaattc 600 aagnattgtt ttccaatttc
ccatccctga tttccagctt tctt 644 2 1115 DNA Homo sapiens 2 gtaggaagtt
acagtaaatg gtagttcatt cttacttaca cacatagcta atcttttttt 60
tttcacttgg aattatgttg aatgtttcat tttgacaaaa aagtagacta gaaggtatgt
120 yctttaagtt gtcttgcatc cattatataa gaaagaaaca ggtgagagga
agagcagaaa 180 gctgagactg gctgatgttc agagcactta ctcctctaga
gggaaagcat gacaccgaac 240 actaagcaca cagctttttg ttgttttggt
tttttctccc gcaaatctta aagtgattcc 300 catgaccttg gccaaggaca
cttcttaaag attaatgact ggcactgaca ttgccccagg 360 cgggccactc
ctcacactgg ctctcagttc ccagccatgc ctggggctca gtcacttcta 420
ttccaccctc tgagactcca ttggtgtcac acaaggtgtc ttcttggctt tgattttgag
480 aatcccctat tttcacttcc agatctgtca gctgccatgg aggaataata
gaaaaccaga 540 aatgcgtgta gagggagatt tctaaaactt cccttgtgtc
gcccatagtt gtagttttgg 600 gttctggcag gtggaacacc ctgaaacctg
gaatcattct atgagaatac agttcagact 660 ttgcagactc cagcccatac
taactgtcat gaagcttgac ttcttgtcat aatgcagcca 720 tcttggagga
aattggccat ttctgcttag atggttggca gggtcgcgct cagctttgct 780
ttctacacta attacatagc attattcaag tattgttttc catttcccat ccctgatttc
840 cagcttctta aagctgactg ttcttgcagg ggccacttgc ttctcctaga
gtacaaaagt 900 aagggccttc cttactaact gcagggtctc tctattacac
ctcaacatac acactttgct 960 gctactgttt gtactgtcta cagtagaatt
tccttatctt gctcctggta gtgcattaca 1020 ggcaagcatg aaatgtaaag
tatttattta aataaaaaga aaacctctaa attggtaatt 1080 gaawwammwm
mmwrwarmww tatagtttgt gacat 1115 3 540 DNA Homo sapiens 3
gggccagaat tcggccgagg cctgcaaacg agaaggctgt ggatttgatt attgtacgaa
60 gtgtctctgt aattatcata ctactaaaga ctgttcagat ggcaagctcc
tcaaagccag 120 ttgtaaaata ggtcccctgc ctggtacaaa gaaaagcaaa
aagaatttac gaagattgtg 180 atctcttatt aaatcaattg ttactgatca
tgaatgttag ttagaaaatg ttaggtttta 240 acttaaaaaa aattgtattg
tgattttcaa ttttatgttg aaatcggtgt agtatcctga 300 ggtttttttc
cccccagaag ataaagagga tagacaacct cttaaaatat ttttacaatt 360
taatgagaaa aagtttaaaa ttctcaatac aaatcaaaca atttaaatat tttaagaaaa
420 aaggaaaagt agatagtgat actgagggta aaaaaaaatt gattcaattt
tatggtaaag 480 gaaacccatg caattttacc tagacagtct taaatatgtc
tggttttcca tctgttagca 540 4 2076 DNA Homo sapiens 4 aggttgctca
gctgcccccg gagcggttcc tccacctgag gcagacacca cctcggttgg 60
catgagccgg cgcccctgca gctgcgccct acggccaccc cgctgctcct gcagcgccag
120 ccccagcgca gtgacagccg ccgggcgccc tcgaccctcg gatagttgta
aagaagaaag 180 ttctaccctt tctgtcaaaa tgaagtgtga ttttaattgt
aaccatgttc attccggact 240 taaactggta aaacctgatg acattggaag
actagtttcc tacacccctg catatctgga 300 aggttcctgt aaagactgca
ttaaagacta tgaaaggctg tcatgtattg ggtcaccgat 360 tgtgagccct
aggattgtac aacttgaaac tgaaagcaag cgcttgcata acaaggaaaa 420
tcaacatgtg caacagacac ttaatagtac aaatgaaata gaagcactag agaccagtag
480 actttatgaa gacagtggct attcctcatt ttctctacaa agtggcctca
gtgaacatga 540 agaaggtagc ctcctggagg agaatttcgg tgacagtcta
caatcctgcc tgctacaaat 600 acaaagccca gaccaatatc ccaacaaaaa
cttgctgcca gttcttcatt ttgaaaaagt 660 ggtttgttca acattaaaaa
agaatgcaaa acgaaatcct aaagtagatc gggagatgct 720 gaaggaaatt
atagccagag gaaattttag actgcagaat ataattggca gaaaaatggg 780
cctagaatgt gtagatattc tcagcgaact ctttcgaagg ggactcagac atgtcttagc
840 aactatttta gcacaactca gtgacatgga cttaatcaat gtgtctaaag
tgagcacaac 900 ttggaagaag atcctagaag atgataaggg ggcattccag
ttgtacagta aagcaataca 960 aagagttacc gaaaacaaca ataaattttc
acctcatgct tcaaccagag aatatgttat 1020 gttcagaacc ccactggctt
ctgttcagaa atcagcagcc cagacttctc tcaaaaaaga 1080 tgctcaaacc
aagttatcca atcaaggtga tcagaaaggt tctacttata gtcgacacaa 1140
tgaattctct gaggttgcca agacattgaa aaagaacgaa agcctcaaag cctgtattcg
1200 ctgtaattca cctgcaaaat atgattgcta tttacaacgg gcaacctgca
aacgagaagg 1260 ctgtggattt gattattgta cgaagtgtct ctgtaattat
catactacta aagactgttc 1320 agatggcaag ctcctcaaag ccagttgtaa
aataggtccc ctgcctggta caaagaaaag 1380 caaaaagaat ttacgaagat
tgtgatctct tattaaatca attgttactg atcatgaatg 1440 ttagttagaa
aatgttaggt tttaacttaa aaaaaattgt attgtgattt tcaattttat 1500
gttgaaatcg gtgtagtatc ctgaggtttt tttcccccca gaagataaag aggatagaca
1560 acctcttaaa atatttttac aatttaatga gaaaaagttt aaaattctca
atacaaatca 1620 aacaatttaa atattttaag aaaaaaggaa aagtagatag
tgatactgag ggtaaaaaaa 1680 aaattgattc aattttatgg taaaggaaac
ccatgcaatt ttacctagac agtcttaaat 1740 atgtctggtt ttccatctgt
tagcatttca gacattttat gttcctctta ctcaattgat 1800 accaacagaa
atatcaactt ctggagtcta ttaaatgtgt tgtcaccttt ctaaagcttt 1860
ttttcattgt gtgtatttcc caagaaagta tcctttgtaa aaacttgctt gttttcctta
1920 tttctgaaat ctgttttaat atttttgtat acatgtaaat atttctgtat
tttttatatg 1980 tcaaagaata tgtctcttgt atgtacatat aaaaataaat
tttgctcaat aaaattgtaa 2040 gcttaaaaaa aaaaaaaaaa aactcgagac tagtgc
2076 5 634 DNA Homo sapiens 5 gggcagaatt cggacgagga cttttcctca
gtgttgacct tagggtgcag ctggatgttt 60 ttaccctcag cggctttcgg
actgtacaga tcctggaagg acaaaagatc ctggctaact 120 gttcttctcc
ctaccaggta gacctgtttg gtatagcaga tttagcacat ttactattgt 180
tcaaggaaca cctacaggtc ttctgggatg ggtccttctg gaaacttagc caaaatattt
240 ctgagctaaa agatggtgaa ttgtggaata aattctttgt gcggattctg
aatgccaatg 300 atgaggccac agtgtctgtt cttggggagc ttgcagcaga
aatgaatggg gtttttgaca 360 ctacattcca aagtcacctg aacaaagcct
tatggaaggt agggaagtta actagtcctg 420 gggctttgct ctttcagtga
gctaggcaat caagtctcac agattgctgc ctcagagcaa 480 tggttgtatt
gtggaacact gaaactgtat gtgctgtaat ttaatttagg acacatttag 540
atgcactacc attgctgttc tactttttgg tacaggtata ttttgacgtc actgatattt
600 tttatacagt gatatactta ctcatggcct tgct 634 6 3725 DNA Homo
sapiens 6 accgttaaat ttgaaacttg gcgggtaggg gtgtgggctt gaggtggccg
gtttgttagg 60 gagtcgtgtg cgtgccttgg tcgcttctgt agctccgagg
gcaggttgcg gaagaaagcc 120 caggcggtct gtggcccaga ggaaaggcct
gcagcaggac gaggacctga gccaggaatg 180 caggatggcg gcggtgaaga
aggaaggggg tgctctgagt gaagccatgt ccctggaggg 240 agatgaatgg
gaactgagta aagaaaatgt acaaccttta aggcaagggc ggatcatgtc 300
cacgcttcag ggagcactgg cacaagaatc tgcctgtaac aatactcttc agcagcagaa
360 acgggcattt gaatatgaaa ttcgatttta cactggaaat gaccctctgg
atgtttggga 420 taggtatatc agctggacag agcagaacta tcctcaaggt
gggaaggaga gtaatatgtc 480 aacgttatta gaaagagctg tagaagcact
acaaggagaa aaacgatatt atagtgatcc 540 tcgatttctc aatctctggc
ttaaattagg gcgtttatgc aatgagcctt tggatatgta 600 cagttacttg
cacaaccaag ggattggtgt ttcacttgct cagttctata tctcatgggc 660
agaagaatat gaagctagag aaaactttag gaaagcagat gcgatatttc aggaagggat
720 tcaacagaag gctgaaccac tagaaagact acagtcccag caccgacaat
tccaagctcg 780 agtgtctcgg caaactctgt tggcacttga gaaagaagaa
gaggaggaag tttttgagtc 840 ttctgtacca caacgaagca cactagctga
actaaagagc aaagggaaaa agacagcaag 900 agctccaatc atccgtgtag
gaggtgctct caaggctcca agccagaaca gaggactcca 960 aaatccattt
cctcaacaga tgcaaaataa tagtagaatt actgtttttg atgaaaatgc 1020
tgatgaggct tctacagcag agttgtctaa gcctacagtc cagccatgga tagcaccccc
1080 catgcccagg gccaaagaga atgagctgca agcaggccct tggaacacag
gcaggtcctt 1140 ggaacacagg cctcgtggca atacagcttc actgatagct
gtacccgctg tgcttcccag 1200 tttcactcca tatgtggaag agactgcaca
acagccagtt atgacaccat gtaaaattga 1260 acctagtata aaccacatcc
taagcaccag aaagcctgga aaggaagaag gagatcctct 1320 acaaagggtt
cagagccatc agcaagcatc tgaggagaag aaagagaaga tgatgtattg 1380
taaggagaag atttatgcag gagtagggga attctccttt gaagaaattc gggctgaagt
1440 tttccggaag aaattaaaag agcaaaggga agccgagcta ttgaccagtg
cagagaagag 1500 agcagaaatg cagaaacaga ttgaagagat ggagaagaag
ctaaaagaaa tccaaactac 1560 tcagcaagaa agaacaggtg atcagcaaga
agagacgatg cctacaaagg agacaactaa 1620 actgcaaatt gcttccgagt
ctcagaaaat accaggaatg actctatcca gttctgtttg 1680 tcaagtaaac
tgttgtgcca gagaaacttc acttgcggag aacatttggc aggaacaacc 1740
tcattctaaa ggtcccagtg tacctttctc catttttgat gagtttcttc tttcagaaaa
1800 gaagaataaa agtcctcctg cagatccccc acgagtttta gctcaacgaa
gaccccttgc 1860 agttctcaaa acctcagaaa gcatcacctc aaatgaagat
gtgtctccag atgtttgtga 1920 tgaatttaca ggaattgaac ccttgagcga
ggatgccatt atcacaggct tcagaaatgt 1980 aacaatttgt cctaacccag
aagacacttg tgactttgcc agagcagctc gttttgtatc 2040 cactcctttt
catgagataa tgtccttgaa ggatctccct tctgatcctg agagactgtt 2100
accggaagaa gatctagatg taaagacctc tgaggaccag cagacagctt gtggcactat
2160 ctacagtcag actctcagca tcaagaagct gagcccaatt attgaagaca
gtcgtgaagc 2220 cacacactcc tctggcttct ctggttcttc tgcctcggtt
gcaagcacct cctccatcaa 2280 atgtcttcaa attcctgaga aactagaact
tactaatgag acttcagaaa accctactca 2340 gtcaccatgg tgttcacagt
atcgcagaca gctactgaag tccctaccag agttaagtgc 2400 ctctgcagag
ttgtgtatag aagacagacc aatgcctaag ttggaaattg agaaggaaat 2460
tgaattaggt aatgaggatt actgcattaa acgagaatac ctaatatgtg aagattacaa
2520 gttattttgg gtggcgccaa gaaactttgc agaattaaca gtaataaagg
tatcttctca 2580 acctgtccca tgggactttt atatcaacct caagttaaag
gaacgtttaa atgaagattt 2640 tgatcatttt tgcagctgtt atcaatatca
agatggctgt attgtttggc accaatatat 2700 aaactgcttc acccttcagg
atcttctcca acacagtgaa tatattaccc atgaaataac 2760 agtgttgatt
atttataacc ttttgacaat agtggagatg ctacacaaag cagaaatagt 2820
ccatggtgac ttgagtccaa ggtgtctgat tctcagaaac agaatccacg atccctatga
2880 ttgtaacaag aacaatcaag ctttgaagat agtggacttt tcctacagtg
ttgaccttag 2940 ggtgcagctg gatgttttta ccctcagcgg ctttcggact
gtacagatcc tggaaggaca 3000 aaagatcctg gctaactgtt cttctcccta
ccaggtagac ctgtttggta tagcagattt 3060 agcacattta ctattgttca
aggaacacct acaggtcttc tgggatgggt ccttctggaa 3120 acttagccaa
aatatttctg agctaaaaga tggtgaattg tggaataaat tctttgtgcg 3180
gattctgaat gccaatgatg aggccacagt gtctgttctt ggggagcttg cagcagaaat
3240 gaatggggtt tttgacacta cattccaaag tcacctgaac aaagccttat
ggaaggtagg 3300 gaagttaact agtcctgggg ctttgctctt tcagtgagct
aggcaatcaa gtctcacaga 3360 ttgctgcctc agagcaatgg ttgtattgtg
gaacactgaa actgtatgtg ctgtaattta 3420 atttaggaca catttagatg
cactaccatt gctgttctac tttttggtac aggtatattt 3480 tgacgtcact
gatatttttt atacagtgat atacttactc atggccttgt ctaacttttg 3540
tgaagaacta ttttattcta aacagactca ttacaaatgg ttaccttgtt atttaaccca
3600 tttgtctcta cttttccctg tacttttccc atttgtaatt tgtaaaatgt
tctcttatga 3660 tcaccatgta ttttgtaaat aataaaatag tatctgttaa
aaaaaaaaaa aaaaaaaaaa 3720 aaaaa 3725 7 567 DNA Homo sapiens
misc_feature (1)...(567) n = A,T,C or G 7 ggccaagaat tcggcacgag
gacaacatac taaagaggcg aggcaatgac tgttggccag 60 ttctcaccgg
ggaaaaaccc actgttagga tggcatgaac atttccttag atcgtggnca 120
gctccgagga atgtggcgtn caggctcttt gagagccatg ggctgcaccc ggccgtaggc
180 tagtgtaact cgcatcccat tgcagtgccg tttcttgact gtgttgctgt
ctcttagatt 240 aaccgtgctg aggctccaca tagctcctgg acctgtgtct
agtacatact gaagcgatgg 300 tcagagtgtg tagagtgaag ttgctgtgcc
cacattgttt gaactcgcgt accccgtaga 360 tacattgtgc aacgttcttc
tgttattccc ttgaggtggt aacttcgtat gttcagttta 420 tgcgatgatt
gttgtaaatg caatgccgta gtttggatta ataagtggat ggtttttgtt 480
tctaaaaaga aaaaaaaaat cagtgttcac ccttatagag acatagtcaa gttcatgttg
540 ataataatca aaggaattac tctcttc 567 8 1365 DNA Homo sapiens 8
acttcatgaa cacggacaat ttcacctccc accgtctccc ccacccctgg tcgggcacgg
60 ggcaggtggt ctacaacggt tctatctact ttaacaagtt ccagagccac
atcatcatca 120 ggtttgacct gaagacagag accatcctca agacccgcag
cctggactat gccggttaca 180 acaacatgta ccactacgcc tggggtggcc
actcggacat cgacctcatg gtggacgaga 240 gcgggctgtg ggccgtgtac
gccaccaacc agaacgctgg caacatcgtg gtcagtaggc 300 tggaccccgt
gtccctgcag accctgcaga cctggaacac gagctacccc aagcgcagcg 360
ccggggaggc cttcatcatc tgcggcacgc tgtacgtcac caacggctac tcagggggta
420 ccaaggtcca ctatgcatac cagaccaatg cctccaccta tgaatacatc
gacatcccat 480 tccagaacaa atactcccac atctccatgc tggactacaa
ccccaaggac cgggccctgt 540 atgcctggaa caacggccac cagatcctct
acaacgtgac cctcttccac gtcatccgct 600 ccgacgagtt gtagctccct
cctcctggaa gccaagggcc cacgtcctca ccacaaaggg 660 actcctgtga
aactgctgcc aaaaagatac caataacact aacaataccg atcttgaaaa 720
atcatcagca gtgcggattc tgacatcgag ggatggcatt acctccgtgt ttctcccttt
780 cgagccggcg ggccacagac gtcggaagaa actcccgtat ttgcagctgg
aactgcagcc 840 cacggcgccc cggttttcct ccccgccctg tccctctctg
gtcaaacaac atactaaaga 900 ggcgaggcaa tgactgttgg ccagttctca
ccggggaaaa acccactgtt aggatggcat 960 gaacatttcc ttagatcgtg
gtcagctccg aggaatgtgg cgtccaggct ctttgagagc 1020 catgggctgc
acccggccgt aggctagtgt aactcgcatc ccattgcagt gccgtttctt 1080
gactgtgttg ctgtctctta gattaaccgt gctgaggctc cacatagctc ctggacctgt
1140 gtctagtaca tactgaagcg atggtcagag tgtgtagagt gaagttgctg
tgcccacatt 1200 gtttgaactc gcgtaccccg tagatacatt gtgcaacgtt
cttctgttat tcccttgagg 1260 tggtaacttc gtatgttcag tttatgcgat
gattgttgta aatgcaatgc cgtagtttgg 1320 attaataagt ggatggtttt
tgtttctaaa aaaaaaaaaa aaaaa 1365 9 1196 DNA Homo sapiens 9
ctcagctcta ggggaatgaa ggctgttttg ctggctgata ctgaaataga ccttttctct
60 acagacatcc ctcctaccaa cgcagtggac ttcactggaa gatgctattt
caccaaaatc 120 tgcaaatgta aactgaagga catcgcatgt ttaaaatgtg
ggaacattgt agtttatcat 180 gtgattgttc catgtagttc ctgtcttctt
tcctgcaaca acagacactt ctggatgttt 240 cacagccagg cagtttatga
tattaacaga ctagactcca caggtgtaaa cgtcctactt 300 cggggcaact
tgccagagat agaagagagt acagatgaag atgtgttaaa tatctcagca 360
gaggagtgta ttagataaat ggaattatga tatatatgat atacaaactt ttttctattt
420 aaaaatatat taatggatca actttaaaat tgttagttgc cagtgatctt
ttttggaaaa 480 caaaaatggg gcatttgttg atttatttat tttctgtctc
taattagtta cctcagtttg 540 attgaagcca gtggagttgt gcttttcctc
tacttctact tcctctcccc cacctttttc 600 tgcccagtgt aggtgtattc
ttaaattcag acgggaagat tctttcacat atcactcagt 660 tacctcccaa
tctgggggag tttttcttac aacttgatac cagataccat taattttaca 720
ttcctgaata aaggcctagt acccacgcat atttcaacca tgcatatatc aagttcaacy
780 gagttttaat aggggattaa aaaaacaagc tgttaggttt ccatgggcac
tggttctcat 840 aggttctatt ggtgataact gctttaacat ggagcaagag
tttgtgaatc aggaaataga 900 ataaattaaa atttaaaata tatagaggaa
tcctcttgat tgctcagcat gatgttagat 960 aaatgagttt gtcagaaaat
atcagtatac gctgtttacc aatgttattt atttacattc 1020 ttctaaagcc
attatggata ttgtattatg agagctaaac ctaaataagt tatcctgttc 1080
cctaggacct tctctgtaaa tagtgaattt tagacgagta gtctgtccta aatcttaaat
1140 agaaaaaaaa actaaagcga tttgcttaag ccattgtaca ttataaagag ctgttt
1196 10 1424 DNA Homo sapiens 10 ctcagctcta ggggaatgaa ggctgttttg
ctggctgata ctgaaataga ccttttctct 60 acagacatcc ctcctaccaa
cgcagtggac ttcactggaa gatgctattt caccaaaatc 120 tgcaaatgta
aactgaagga catcgcatgt ttaaaatgtg ggaacattgt agkttatcat 180
gtgattgttc catgtagttc ctgtcttctt tcctgcaaca acagacactt ctggatgttt
240 cacagccagg cagtttatga tattaacaga ctagactcca caggtgtaaa
cgtcctactt 300 cggggcaact tgccagagat agaagagagt acagatgaag
atgtgttaaa tatctcagca 360 gaggagtgta ttagataaat ggaattatga
tatatatgat atacaaactt ttttctattt 420 aaaaatatat taatggatca
actttaaaat tgttagttgc cagtgatctt tttkggaaaa 480 caaaaatggg
gcatttgttg atttatttat tttctgtctc taattagtta cctcagtttg 540
attgaagcca gtggagttgt gcttttcctc tacttctact tcctctcccc cacctttttc
600 tgcccagtgt aggtgtattc ttaaattcag acgggaagat tctttcacat
atcactcagt 660 tacctcccaa tctgggggag tttttcttac aacttgatac
cagataccat taattttaca 720 ttcctgaata aaggcctagt acccacgcat
atttcaacca tgcatatatc aagttcaacy 780 gagttttaat aggggattaa
aaaaacaagc tgttaggttt ccatgggcac tggttctcat 840 aggttctatt
ggtgataact gctttaacat ggagcaagag tttgtgaatc aggaaataga 900
ataaattaaa atttaaaata tatagaggaa tcctcttgat tgctcagcat gatgttagat
960 aaatgagttt gtcagaaaat atcagtatac gctgtttacc aatgttattt
atttacattc 1020 ttctaaagcc attatggata ttgtattatg agagctaaac
ctaaataagt tatcctgttc 1080 cctaggacct tctctgtaaa tagtgaattt
tagacgagta gtctgtccta aatcttaaat 1140 agaaaaaaaa actaaagcga
tttgcttaag ccattgtaca ttataaagag ctgttttgtt 1200 ttgctttgct
ttgctttgtt ttgttttttt taaagctgca ttcagagcca caaaggaata 1260
ggaaagtagg gtagtgttgg attctggttt tatgtaactc taaaataaat gtatctcttt
1320 aatatctcag ttgtagggat tttgtcaata ccaaagcaga ctgagttgtg
gttttgtaaa 1380 taaagttttt tctaaaaatg aaaaaaaaag aaaaaaaaaa aaaa
1424 11 460 DNA Homo sapiens misc_feature (1)...(460) n = A,T,C or
G 11 agacagngac gtatggaaaa gntcttaaca gatnatttaa atgacctcca
gggtcgcaat 60 gatnatgacg ccagtggcac tngggacttc tatggggaca
ntttgtttgt gaaccagatg 120 atgaaagtgg caaggccaaa caggatncat
ncgcctagag nagaanacna agatgatgat 180 gacgatgcct atagcngatg
tgtttgaatt ngaattttca gagacccccc tcttaccgtg 240 ttataacatc
caagtatctg tggctcaggg gccacgaaac tggctactgc tttcggatgt 300
ccttaagaaa ttganaatgt cctcccgcat atttcgctgc anttttccaa acgnggaaat
360 tgtcaccatt gcagaggcag aattttatcg gtaggtttct gcnagtctct
tgntctcttg 420 ctccaaagac ctggcaagcc ttcaaccctt gaaaggnaan 460 12
2206 DNA Homo sapiens 12 cagaagacag atgtgctgtg tgcagacgaa
gaagaggatt gccaggctgc ctccctgctg 60 cagaaataca ccgacaacag
cgagaagcca tccgggaaga gactgtgcaa aaccaaacac 120 ttgatccctc
aggagtccag gcggggattg ccactgacag gggaatacta cgtggagaat 180
gccgatggca aggtgactgt ccggagattc agaaagcggc cggagcccag ttcggactat
240 gatctgtcac cagccaagca ggagccaaag cccttcgacc gcttgcagca
actgctacca 300 gcctcccagt ccacacagct gccatgctca agttcccctc
aggagaccac ccagtctcgc 360 cctatgccgc cggaagcacg gagacttatt
gtcagtaaga acgctggcga gacccttctg 420 cagcgggcag ccaggcttgg
ctatgaggaa gtggtcctgt actgcttaga gaacaagatt 480 tgtgatgtaa
atcatcggga caacgcaggt tactgcgccc tgcatgaagc ttgtgctagg 540
ggctggctca acattgtgcg acacctcctt gaatatggcg ctgatgtcaa ctgtagtgcc
600 caggatggaa ccaggcctct gcacgatgct gttgagaacg atcacttgga
aattgtccga 660 ctacttctct cttatggtgc tgaccccacc ttggctacgt
actcaggtag aaccatcatg 720 aaaatgaccc acagtgaact tatggaaagg
ttcttaacag attatttaaa tgacctccag 780 ggtcgcaatg atgatgacgc
cagtggcact tgggacttct atggcagctc tgtttgtgaa 840 ccagatgatg
aaagtggcta tgatgtttta gccaaccccc caggaccaga agaccaggat 900
gatgatgacg atgcctatag cgatgtgttt gaatttgaat tttcagagac ccccctctta
960 ccgtgttata acatccaagt atctgtggct caggggtgag catggctgtc
atgtgattga 1020 aaactagctg agctgctctt gaggccacga aactggctac
tgctttcgga tgtccttaag 1080 aaattgaaaa tgtcctcccg catatttcgc
tgcaattttc caaacgtgga aattgtcacc 1140 attgcagagg cagaatttta
tcggcaggtt tctgcaagtc tcttgttctc ttgctccaaa 1200 gacctggaag
ccttcaaccc tgaaagtaag gagctgttag atctggtgga attcacgaac 1260
gaaattcaga ctctgctggg ctcctctgta gagtggctcc accccagtga tctggcctca
1320 gacaactact ggtgagcaag ctggacccac catgtacagt gtgttatagt
gttaatcctt 1380 gtgcatatgt gtcataatac aactatttct gtaaagaaag
gacactatta catatgaaaa 1440 tatctcttct ttatataaga gaaattactc
cagtcagaag gacttagaaa catgtttttt 1500 tccttttaaa cttttaagtc
agtttttatg aagttgttat aatgtttctt tacttttcaa 1560 tgcacacatg
ctttgggata cgtttgtttt tacttggaac atttgtttct tttctttttt 1620
aaggagaaaa aaaaaatgag taaaaggagc tccacacttt gacttaattt catacaaagc
1680 tctgatgaca ggccatgact gtagagtggt cagaactgtg tggttggttt
gagggagcga 1740 attcggggaa ggcacttggt gatataactt tgttttgttt
acagagtacc tgctcgggcc 1800 aggtaaatgc tattggatgt aatccagtag
tgtgtaatat aaattcaaac catatccaca 1860 cacaacaact aattgtatga
aacttttata tcctaattta aaagctgtga aattagtttt 1920 cacgcatcaa
accggattgt ttatatgttt aaacatttta tgctcttatt taaagaagac 1980
tttgagctat ttttttctgt accctgtaaa atattgaaaa ctaacataat atgttgaggt
2040 tgcttggaaa tgtacataaa actaaaattt tctgaatcgt gtgtttatgt
ttgaaatctg 2100 tgttttaact ttgtaagtaa attctctgcc tttgtattta
tattttacaa aattttctta 2160 aaaggcataa aactgttgag gaaaggagaa
aaaaaaaaaa aaaaaa 2206 13 680 DNA Homo sapiens misc_feature
(1)...(680) n = A,T,C or G 13 ataagatccc agctttgcgg gaactcatgc
actatctcag ggaggtgatg caggattacc 60 gagatgagct caaggacttc
tttgcagttg acaaacagct ggcatcagag cttgagtatg 120 acatgaagaa
gtaccaggaa cagctggtcc aggagcagga gctagcaaaa catgcagatg 180
tggccgggac ggctggaggt gctgaggtgg cacctgtggc acaggttgcc ctgtgtttag
240 aaacagtgcc agttcctgct ggccaagaaa accctgccat gtcacctgcc
gtgagccagc 300 cctgcacacc cagggcaagt gctggccatg tagcagtatc
atctcctaca cctgaaacag 360 ggccattgca gaggttgctg cccaaagcca
ggcccatgtc cctgagcacc attgcaatcc 420 tgaattctgt caagaaagcc
gtggagtcaa agagcaggca tcggagtcgg agcttaggag 480 tgctgccttt
cactttaaat tctggaagcc cagaaaaaac gtgcagtcag gtgtcttcat 540
acagtttgga gcaagagtcg aatggcgaga ttgagcacgt gaccaagcgg gccatcagca
600 cccccgagaa gagcatcagt gatgtcacgt tttggagcan gggtcaagtt
acatcgggac 660 accacgggac ttccgtcgtc 680 14 5023 DNA Homo sapiens
14 ggcggcggcg agccggtgcc ctgggatcat ggtggcgttg cggggccttg
gtagcggcct 60 gcagccctgg tgtccgctgg atcttagact cgaatgggtt
gacacagtgt gggaactgga 120 tttcacagag actgagcctt tggatcccag
catagaagca gagatcatag agactggatt 180 ggctgcattc acaaaactct
atgaaagcct tttacccttt gctactggag aacatggatc 240 tatggagagt
atctggacct tcttcattga gaacaatgtt tcccatagta cactggtggc 300
attgttctat cattttgttc aaatagttca taagaagaat gtcagtgtac agtatcgaga
360 atatggcctt catgccgctg ggctttactt tttgctacta gaagtaccag
gcagtgtagc 420 caatcaagta ttccacccag tgatgtttga caaatgcatt
cagactctaa agaagagctg 480 gccccaggaa tctaacttga atcggaaaag
aaagaaagaa cagcctaaga gctctcaggc 540 taaccccggg aggcatagaa
aaaggggaaa gccacccagg agagaagata ttgagatgga 600 tgaaattata
gaagaacaag aagatgagaa tatttgtttt tctgcccggg acctttctca 660
aattcgaaat gccatctttc accttttaaa gaatttttta aggcttctgc caaagttttc
720 cttgaaagaa aagccacaat gtgtacagaa ttgtatagag gtctttgttt
cattaactaa 780 ttttgagcca gttcttcatg aatgtcatgt tacacaagcc
agagctctta accaagcaaa 840 atacatacca gaactggctt attatggatt
gtatttgctg tgctctccca ttcatggaga 900 aggagataag gtcatcagtt
gtgttttcca tcaaatgctc agtgtaatat taatgttaga 960 agttggtgaa
ggatcccatc gtgcccccct tgctgttacc tcccaagtca tcaactgtag 1020
aaaccaggcg gtccagttta tcagcgccct tgtggatgaa ttaaaggaga gtatattccc
1080 agtcgtccgt atcttactgc agcacatctg tgccaaggtg gtagataaat
cagagtatcg 1140 tacttttgca gcccagtccc tagtccagct gctcagtaaa
cttccttgtg gggaatacgc 1200 tatgttcatt gcctggcttt acaaatactc
ccgaagttcc aagatcccac accgggtttt 1260 tactcttgat gttgtcttag
ctctgttaga actgcctgaa agagaggtgg ataacaccct 1320 ctccttggag
catcagaagt tcttaaagca taagttcctg gtgcaggaaa ttatgtttga 1380
tcgttgctta gacaaggcgc ctactgtccg cagcaaggca ctgtccagct ttgcacactg
1440 tctggagttg actgttacca gtgcgtcgga gagtatcctg gagctcctga
ttaacagtcc 1500 tacgttttct gtaatagaga gtcaccctgg taccttactg
agaaattcat cagctttttc 1560 ctaccaaagg cagacatcta accgttccga
accctcaggg gagatcaaca tagacagcag 1620 tggtgaaaca gttggatctg
gagaaagatg tgtcatggca atgctgagaa ggaggatcag 1680 ggatgagaag
accaacgtta ggaagtctgc actgcaggta ttagtgagta ttttgaaaca 1740
ctgtgatgtc tcaggcatga aggaagacct gtggattctg caggaccagt gtcgggaccc
1800 tgcagtgtct gtccggaagc aggccctcca gtctcttact gaactcctta
tggctcagcc 1860 tagatgcgtg cagatccaga aagcctggtt gcggggggtg
gtcccggtgg tgatggactg 1920 cgagagcact gtgcaggaga aggccctgga
gttcctggac cagctgctgc tgcagaacat 1980 ccggcatcac agtcattttc
actctgggga cgacagccag gtcctcgcct gggcgcttct 2040 tactctcctc
accaccgaaa gccaggaact gagccgatat ttaaataagg cttttcatat 2100
ctggtccaag aaagaaaaat tctcacccac ttttataaac aatgtaatat ctcacactgg
2160 cacggaacat tcggcacctg cctggatgct gctctccaag attgctggct
cctcacccag 2220 gctggactac agcagaataa tacaatcttg ggagaaaatc
agcagtcagc agaatcccaa 2280 ttcaaacacc ttaggacata ttctctgtgt
gattgggcat attgcaaagc atcttcctaa 2340 gagcacccgg gacaaagtga
ctgatgctgt caagtgtaag ctgaatggat ttcagtggtc 2400 tctagaggtg
atcagttcag ctgttgacgc cttgcagagg ctttgtagag catctgcaga 2460
gacaccagca gaggagcagg aattgctgac gcaggtgtgt ggggatgtac tctccacctg
2520 cgagcaccgc ctctccaaca tcgttctcaa ggagaatgga acagggaata
tggacgaaga 2580 cctgttggtg aagtacattt ttaccttagg ggatatagcc
cagctgtgtc cagccagggt 2640 ggagaagcgc atcttccttc tgattcagtc
cgtcctggct tcgtctgctg atgctgacca 2700 ctcaccatca tctcaaggca
gcagtgaggc cccagcgtct cagccacccc cccaggtcag 2760 aggttctgtc
atgccctctg tgattagagc acatgccatc attaccttag gtaagctgtg 2820
cttacagcac gaggatctgg caaagaagag catcccagcc ctggtgcgag agctcgaggt
2880 gtgtgaggac gtggctgtcc gcaacaacgt catcattgta atgtgcgatc
tctgcattcg 2940 ctacaccatc atggtggaca agtatattcc caacatctcc
atgtgtctga aggattccga 3000 cccattcatc cggaagcaga cactcatctt
gcttaccaat ctcttgcagg aggaatttgt 3060 gaaatggaag ggctccctgt
tcttccgatt tgtcagcact ctgatcgatt cacacccaga 3120 cattgccagc
ttcggggagt tttgcctggc tcacctgtta ctgaagagga accctgtcat 3180
gttcttccaa cacttcattg aatgtatttt tcactttaat aactatgaga agcatgagaa
3240 gtacaacaag ttcccccagt cagagagaga gaagcggctg ttttcattga
agggaaagtc 3300 aaacaaagag agacgaatga aaatctacaa atttcttcta
gagcacttca cagatgaaca 3360 gcgattcaac atcacttcca aaatctgcct
tagtattttg gcgtgctttg ctgatggcat 3420 cctacccctg gacctggacg
ccagtgagtt actctcagac acgtttgagg tcctcagctc 3480 aaaggagatc
aagcttttgg caatgagatc taaaccagac aaagacctcc ttatggaaga 3540
agatgacatg gccttggcaa atgtagtcat gcaggaagct cagaagaagc tcatctcaca
3600 agttcagaag aggaatttca tagaaaatat tattccaatt atcatctccc
tgaagactgt 3660 gctggagaaa aataagatcc cagctttgcg ggaactcatg
cactatctca gggaggtgat 3720 gcaggattac cgagatgagc tcaaggactt
ctttgcagtt gacaaacagc tggcatcaga 3780 gcttgagtat gacatgaaga
agtaccagga acagctggtc caggagcagg agctagcaaa 3840 acatgcagat
gtggccggga cggctggagg tgctgaggtg gcacctgtgg cacaggttgc 3900
cctgtgttta gaaacagtgc cagttcctgc tggccaagaa aaccctgcca tgtcacctgc
3960 cgtgagccag ccctgcacac ccagggcaag tgctggccat gtagcagtat
catctcctac 4020 acctgaaaca gggccattgc agaggttgct gcccaaagcc
aggcccatgt ccctgagcac 4080 cattgcaatc ctgaattctg tcaagaaagc
cgtggagtca aagagcaggc atcggagtcg 4140 gagcttagga gtgctgcctt
tcactttaaa ttctggaagc ccagaaaaaa cgtgcagtca 4200 ggtgtcttca
tacagtttgg agcaagagtc gaatggcgag attgagcacg tgaccaagcg 4260
ggccatcagc acccccgaga agagcatcag tgatgtcacg tttggagcag gggtcagtta
4320 catcgggaca ccacggactc cgtcgtcagc caaagagaaa attgaaggcc
ggagtcaagg 4380 aaatgacatc ttatgtttat cactgcctga taaaccgccc
ccacagcctc agcagtggaa 4440 tgtgcggtct cccgccagga ataaagacac
tccagcctgc agcaggaggt ccctccgaaa 4500 gacccctctg aaaacagcca
actaaacagc gcctcccacc agtgtccagg caggcaggag 4560 cccttgagga
agcagtctcg tgtcctccgt gtgaaggcag ctggatcact tcccgcagtc 4620
cttgggcagc gctttgctgt ggaacacgag agctcctcct caggggcctg gcactcacct
4680 tctattctgt atgatgtatt tggttaaaca ctgtcaaata atagagatgt
gccagattta 4740 gattttctta ccctaatctg tttaatattg taactttatt
ccatttgaaa gtgtcaagcc 4800 cattcagata agctataatc tggtctttaa
ggaatacaac tttaaaactg cagctttctt 4860 ttatataaat caagcctctg
ttaacttgaa ttccttatag tacatatttt cccatctgta 4920 atgccggaat
tttgattcta atattttttc tattatttat aagtgcaaat ttttttaaaa 4980
agtgtacagc tttcttaaag taataaaggt ttagcataaa tac 5023 15 403 DNA
Homo sapiens 15 ccatcacggg gaattctgct gctgttatta ccccattcaa
gttgacaact gaggcaacgc 60 agactccagt ctccaataag aaaccagtgt
ttgatcttaa agcaagtttg tctcgtcccc 120 tcaactatga accacacaaa
ggaaagctaa aaccatgggg gcaatctaaa gaaaataatt 180 atctaaatca
acatgtcaac agaattaact tctacaagaa aacttacaaa caaccccatc 240
tccagacaaa ggaagagcaa cggaagaaac gcgagcaaga acgaaaggag aagaaagcaa
300 aggttttggg aatgcgaagg ggcctcattt tggctgaaga ttaataattt
tttaacatct 360 tgtaaatatt cctgtattct caactttttt ccttttgtaa att 403
16 890 DNA Homo sapiens misc_feature (1)...(890) n = A,T,C or G 16
agcataagcg tntcactgac caagactcca gccagaaagt ctgcacatgt gaccgtgtct
60 gggggcaccc aaaaaggcga ggctgtgctt gggacacaca aattaaagac
catcacgggg 120 aattctgctg ctgttattac cccattcaag ttgacaactg
aggcaacgca gactccagtc 180 tccaataaga aaccagtgtt tgatcttaaa
gcaagtttgt ctcgtcccct caactatgaa 240 ccacacaaag gaaagctaaa
accatggggg caatctaaag aaaataatta tctaaatcaa 300 catgtcaaca
gaattaactt ctacaagaaa acttacaaac aaccccatct ccagacaaag 360
gaagagcaac ggaagaaacg cgagcaagaa cgaaaggaga agaaagcaaa ggttttggga
420 atgcgaaggg gcctcatttt ggctgaagat taataatttt ttaacatctt
gtaaatattc 480 ctgtattctc aacttttttc cttttgtaaa tttttttttt
tttgctgtca tccccacttt 540 agtcacgaga tctttttctg ctaactgttc
atagtctgtg gtagtgtcca tgggttcttc 600 atgtgctatg atctctgaaa
agacgttatc accttaaagc tcaaattctt tgggatggtt 660 tttacttaag
tccattaaca attcaggttt ctaacgagac ccatcctaaa attctgtttc 720
tagattttta atgtcaagtt cccaagttyc ccctgctggt tctaatatta acagaactgc
780 agtcttctgc tagccaatag catttacctg atggcagcta gttatgccag
ctttagggag 840 aatttgaaca ttttccagga atgggggaag ctgggaaaga
aaggccacct 890 17 371 DNA Homo sapiens 17 ttggctcagc aggacaatat
ggtgggaaat gacaaagtaa ctcctgtggc cctaggtcag 60 gttctcttga
ggaaaacaaa aaggctggaa tgatacagct cttcgtaaac caggtgcctc 120
cagtgcctgc ggttattccc aagtccacat tttgcagaca gggccctaaa atgtctagct
180 aggaagttcc tgagcctgtt tttttaaaat tctacacaca cacatgcaca
cacacacgca 240 cgtgtgcaca catgcggata tatacatcct caccttttct
tgagattact gctcagaaga 300 aggcacattt ggtttggtct gcttaccagg
tgctgaagtg ggagcggccg caagcttawt 360 tccttttagt g 371 18 376 DNA
Homo sapiens 18 attctttggc tcagcaggac aatatggtgg gaaatgacaa
agtaactcct gtggccctag 60 gtcaggttct cttgaggaaa acaaaaaggc
tggaatgata cagctcttcg taaaccaggt 120 gcctccagtg cctgcggtta
ttcccaagtc cacattttgc agacagggcc ctaaaatgtc 180 tagctaggaa
gttcctgagc ctgttttttt aaaattctac acacacacat gcacacacac 240
acgcacgtgt gcacacatgc ggatatatac atcctcacct tttcttgaga ttactgctca
300 gaagaaggca catttggttt ggtctgctta ccaggtgctg aagtgggagc
ggccgcaagc 360 ttawttcctt ttagtg 376 19 512 DNA Homo sapiens
misc_feature (1)...(512) n = A,T,C or G 19 ccatgtgata ctgtatgaac
ctangtagnt tggaagaaaa agtagggttt ttgtatacta 60 gcttttgtat
ttgaattaat tatcattcca gctttttata tactatattt catttatgaa 120
gaaattgatt ttcttttggg agncactttt aatctgtaan tttaaaatac aagtctgaat
180 atttatagtt gattcttaac tgtgcatana cctagatata ccattatccc
ttttatacct 240 aanaagggca tgctaataat taccactgtc aaagaggcaa
aggnggtgat ttttgnntat 300 gaagttaagc ctcagnggag gctcatttgt
tagtttttag cngganctaa ngntaaactc 360 agggtnccct gagctatatg
cacactcaga cctctttgct ttacccagng gcgttngtga 420 gttgctcagc
agtacaaact gcccttacct gacagagccc tgnctttgac ctgctcagcc 480
ctgtgcgcta atcctctagt agcccaatca na 512 20 3410 DNA Homo sapiens 20
gcaccaggcg cccagtggag ccgtttggga gaattgcctg cgccacgcag cggggccgga
60 caggcggtaa ggatctgatt aggctttcga acttgagttt gactgatgtc
ttctgtgtgg 120 tgtccgctaa atcccacagc atataggatc agtcgcattg
gttataaggt ttgcttctgg 180 ctgggtgcgg tggctcatgc ctgtaatcca
acattgggag gccaaggcag gcggaccacc 240 tgaagtcggg agcttgagtc
cagccactgt ctgggtactg ccagccatcg ggcccaggtc 300 tctggggttg
tcttaccgca gtgagtacca cgcggtacta cagagaccgg ctgcccgtgt 360
gcccggcagg tggagccgcc gcatcagcgg cctcggggaa tggaagcgga gaacgcgggc
420 agctattccc ttcagcaagc tcaagctttt tatacgtttc catttcaaca
actgatggct 480 gaagctccta atatggcagt tgtgaatgaa cagcaaatgc
cagaagaagt tccagcccca 540 gctcctgctc aggaaccagt gcaagaggct
ccaaaaggaa gaaaaagaaa acccagaaca 600 acagaaccaa aacaaccagt
ggaacccaaa aaacctgttg agtcaaaaaa atctggcaag 660 tctgcaaaac
caaaagaaaa acaagaaaaa attacagaca catttaaagt aaaaagaaaa 720
gtagaccgtt ttaatggtgt ttcagaagct gaacttctga ccaagactct ccccgatatt
780 ttgaccttca atctggacat tgtcattatt ggcataaacc cgggactaat
ggctgcttac 840 aaagggcatc attaccctgg acctggaaac catttttgga
agtgtttgtt tatgtcaggg 900 ctcagtgagg tccagctgaa ccatatggat
gatcacactc taccagggaa gtatggtatt 960 ggatttacca acatggtgga
aaggaccacg cccggcagca aagatctctc cagtaaagaa 1020 tttcgtgaag
gaggacgtat tctagtacag aaattacaga aatatcagcc acgaatagca 1080
gtgtttaatg gaaaatgtat ttatgaaatt tttagtaaag aagtttttgg agtaaaggtt
1140 aagaacttgg aatttgggct tcagccccat aagattccag acacagaaac
tctctgctat 1200 gttatgccat catccagtgc aagatgtgct cagtttcctc
gagcccaaga caaagttcat 1260 tactacataa aactgaagga cttaagagat
cagttgaaag gcattgaacg aaatatggac 1320 gttcaagagg tgcaatatac
atttgaccta cagcttgccc aagaggatgc aaagaagatg 1380 gctgttaagg
aagaaaaata tgatccaggt tatgaggcag catatggtgg tgcttacgga 1440
gaaaatccat gcagcagtga accttgtggc ttctcttcaa atgggctaat tgagagcgtg
1500 gagttaagag gagaatcagc tttcagtggc attcctaatg ggcagtggat
gacccagtca 1560 tttacagacc aaattccttc ctttagtaat cactgtggaa
cacaagaaca ggaagaagaa 1620 agccatgctt aagaatggtg cttctcagct
ctgcttaaat gctgcagttt taatgcagtt 1680 gtcaacaagt agaacctcag
tttgctaact gaagtgtttt attagtattt tactctagtg 1740 gtgtaattgt
aatgtagaac agttgtgtgg tagtgtgaac cgtatgaacc taagtagttt 1800
ggaagaaaaa gtagggtttt tgtatactag cttttgtatt tgaattaatt atcattccag
1860 ctttttatat actatatttc atttatgaag aaattgattt tcttttggga
gtcactttta 1920 atctgtaatt ttaaaataca agtctgaata tttatagttg
attcttaact gtgcataaac 1980 ctagatatac cattatccct tttataccta
agaagggcat gctaataatt accactgtca 2040 aagaggcaaa ggtgttgatt
tttgtatata agttaagcct cagtggagtc tcatttgtta 2100 gtttttagtg
gtaactaagg gtaaactcag ggttccctga gctatatgca cactcagacc 2160
tctttgcttt accagtggtg tttgtgagtt gctcagtagt aaaaactggc ccttacctga
2220 cagagccctg gctttgacct gctcagccct gtgtgttaat cctctagtag
ccaattaact 2280 actctggggt ggcaggttcc agagaatcga gtagaccttt
tgccactcat ctgtgtttta 2340 cttgagacat gtaaatatga tagggaagga
actgaatttc tccattcata tttataacca 2400 ttctagtttt atcttccttg
gctttaagag tgtgccatgg aaagtgataa gaaatgaact 2460 tctaggctaa
gcaaaaagat gctggagata tttgatactc tcatttaaac tggtgcttta 2520
tgtacatgag atgtactaaa ataagtaata tagaattttt cttgctaggt aaatccagta
2580 agccaataat tttaaagatt ctttatctgc atcattgctg tttgttacta
taaattaaat 2640 gaacctcatg gaaaggttga ggtgtatacc tttgtgattt
tctaatgagt tttccatggt 2700 gctacaaata atccagacta ccaggtctgg
tagatattaa agctgggtac taagaaatgt 2760 tatttgcatc ctctcagtta
ctcctgaata ttctgatttc atacgtaccc agggagcatg 2820 ctgttttgtc
aatcaatata aaatatttat gaggtctccc ccacccccag gaggttatat 2880
gattgctctt ctctttataa taagagaaac aaattcttat tgtgaatctt aacatgcttt
2940 ttagctgtgg ctatgatgga ttttattttt tcctaggtca agctgtgtaa
aagtcattta 3000 tgttatttaa atgatgtact gtactgctgt ttacatggac
gttttgtgcg ggtgctttga 3060 agtgccttgc atcagggatt aggagcaatt
aaattatttt ttcacgggac tgtgtaaagc 3120 atgtaactag gtattgcttt
ggtatataac tattgtagct ttacaagaga ttgttttatt 3180 tgaatgggga
aaataccctt taaattatga cggacatcca ctagagatgg gtttgaggat 3240
tttccaagcg tgtaataatg atgtttttcc taacatgaca gatgagtagt aaatgttgat
3300 atatcctata catgacagtg tgagactttt tcattaaata atattgaaag
attttaaaat 3360 tcatttgaaa gtctgatggc ttttacaata aaagatatta
agaattgtta 3410 21 627 DNA Homo sapiens 21 ggccaagaat tcggccgagg
ggtgccgcgg ccatggagaa gcttagctcc atcaaatctc 60 aaacaattta
tgagattatt gataattctc aaggattcta cgtttgtcca gtggagcccc 120
aaaatagaag caagatgaat attccattcc gcattggcaa tgccaaagga gatgatgctt
180 tagaaaaaag atttcttgat aaagctcttg aactcaatat gttgtccttg
aaagggcata 240 ggtctgtggg aggcatccgg gcctctctgt ataatgctgt
cacaattgaa gacgttcaga 300 agctggccgc cttcatgaaa aaatttttgg
agatgcatca gctatgaaca catcctaacc 360 aggatatact ctgttcttga
acaacataca aagtttaaag taacttgggg atggctacaa 420 aaagttaaca
cagtattttt ctcaaatgaa catgtttatt gcagattctt cttttttgaa 480
agaacaacag caaaacatcc acaactctgt aaagctggtg ggacctaatg tcaccttaat
540 tctgacttga actggaagca ttttaagaaa tcttgttgct tttctaacaa
attcccgcgt 600 attttgcctt tgctgctctt tttctag 627 22 1065 DNA Homo
sapiens 22 ccttggctga ctcaccgccc tcgccgccgc accatggacg cccccaggca
ggtggtcaac 60 tttgggcctg gtcccgccaa gctgccgcac tcagtgttgt
tagagataca aaaggaatta 120 ttagactaca aaggagttgg cattagtgtt
cttgaaatga gtcacaggtc atcagatttt 180 gccaagatta ttaacaatac
agagaatctt gtgcgggaat tgctagctgt tccagacaac 240 tataaggtga
tttttctgca aggaggtggg tgcggccagt tcagtgctgt ccccttaaac 300
ctcattggct tgaaagcagg aaggtgtgcg gactatgtgg tgacaggagc ttggtcagct
360 aaggccgcag aagaagccaa gaagtttggg actataaata tcgttcaccc
taaacttggg 420 agttatacaa aaattccaga tccaagcacc tggaacctca
acccagatgc ctcctacgtg 480 tattattgcg caaatgagac ggtgcatggt
gtggagtttg actttatacc cgatgtcaag 540 ggagcagtac tggtttgtga
catgtcctca aacttcctgt ccaagccagt
ggatgtttcc 600 aagtttggtg tgatttttgc tggtgcccag aagaatgttg
gctctgctgg ggtcaccgtg 660 gtgattgtcc gtgatgacct gctggggttt
gccctccgag agtgcccctc ggtcctggaa 720 tacaaggtgc aggctggaaa
cagctccttg tacaacacgc ctccatgttt cagcatctac 780 gtcatgggct
tggttctgga gtggattaaa aacaatggag gtgccgcggc catggagaag 840
cttagctcca tcaaatctca aacaatttat gagattattg ataattctca aggattctac
900 gtgtctgtgg gaggcatccg ggcctctctg tataatgctg tcacaattga
agacgttcag 960 aagctggccg ccttcatgaa aaaatttttg gagatgcatc
agctatgaac acatcctaac 1020 caggatatac tctgttcttg aacaacatac
aaagtttaaa gtaac 1065 23 578 DNA Homo sapiens misc_feature
(1)...(578) n = A,T,C or G 23 gcctcgggcc aagaattcgg cacgaggcca
agttaaggaa cttgaagcta atgtacttgc 60 tacagcccct gacaaaaaaa
gcagaaattg ctagaagaaa acgttagtgc tttcaaaaca 120 gaatangang
ctgnggctga gaaagctggt aaagtagaag ctgaggttaa acgcttacac 180
aataccatcg tagaaatcaa taatcataaa ctcaaggccc aacaagacaa acttgataaa
240 ataaataagc aattagatga atgtgcttct gctattacta aagcccaagt
agcaatcaag 300 actgctgaca gaaaccttca aaaggcacaa gactctgtct
tgcgtacaga gaaagaaata 360 aaagatactg agaaagaggt ggatgaccta
acagcagagc tgaaaagtct tgaggacaaa 420 gcagcagagg tcgtaaagaa
tacaaatgct gcagagcagt tcttttcggt gtttaggaat 480 ccttaccaga
gatccagaaa gaacatcgca atctgcttca agaattaaaa gttattcaag 540
aaaatgaaca tgctcttcaa aaagatgcct tagtatta 578 24 3799 DNA Homo
sapiens 24 atagtaaacc agaacttcaa atcctatgct ggggagaaaa ttctgggacc
tttccataag 60 cgcttttcct gtattatcgg gccaaatggc agtggcaaat
ccaatgttat tgattctatg 120 ctttttgtgt ttggctatcg agcacaaaaa
ataagatcta aaaaactctc agtattaata 180 cataattctg atgaacacaa
ggacattcag agttgtacag tagaagttca ttttcaaaag 240 ataattgata
aggaagggga tgattatgaa gtcattccta acagtaattt ctatgtatcc 300
agaacggcct gcagagataa tacttctgtc tatcacataa gtggaaagaa aaagacattt
360 aaggatgttg gaaatcttct tcgaagccat ggaattgact tggaccataa
tagattttta 420 attttacagg gtgaagttga acaaattgct atgatgaaac
caaaaggcca gactgaacac 480 gatgagggta tgcttgaata tttagaagat
ataattggtt gtggacggct aaatgaacct 540 attaaagtct tgtgtcaaag
agttgaaata ttaaatgaac acagaggaga gaagttaaac 600 agggtaaaga
tggtggaaaa ggaaaaggat gccttagaag gagagaaaaa catagctatc 660
gaatttctta ccttggaaaa tgaaatattt agaaaaaaga atcatgtttg tcaatattat
720 atttatgagt tgcagaaacg aattgctgaa atggaaactc aaaaggaaaa
aattcatgaa 780 gataccaaag aaattaatga gaagagcaat atactatcaa
atgaaatgaa agctaagaat 840 aaagatgtaa aagatacaga aaagaaactg
aataaaatta caaaatttat tgaggagaat 900 aaagaaaaat ttacacacgt
agatttggaa gatgttcaag ttagagaaaa gttaaaacat 960 gccacgagta
aagccaaaaa actggagaaa caacttcaaa aagataaaga aaaggttgaa 1020
gaatttaaaa gtatacctgc caagagtaac aatatcatta atgaaacaac aaccagaaac
1080 aatgccctcg agaaggaaaa agagaaagaa gaaaaaaaat taaaggaagt
tatggatagc 1140 cttaaacagg aaacacaagg gcttcagaaa gaaaaagaaa
gtcgagagaa agaacttatg 1200 ggtttcagca aatcggtaaa tgaagcacgt
tcaaagatgg atgtagccca gtcagaactt 1260 gatatctatc tcagtcgtca
taatactgca gtgtctcaat taactaaggc taaggaagct 1320 ctaattgcag
cttctgagac tctcaaagaa aggaaagctg caatcagaga tatagaagga 1380
aaactccctc aaactgaaca agaattaaag gagaaagaaa aagaacttca aaaacttaca
1440 caagaagaaa caaactttaa aagtttggtt catgatctct ttcaaaaagt
tgaagaagca 1500 aagagctcat tagcaatgaa ttcgagtagg gggaaagtcc
ttgatgcaat aattcaagaa 1560 aaaaaatctg gcaggattcc aggaatatat
ggaagattgg gggacttagg agccattgat 1620 gaaaaatacg acgtggctat
atcatcctgt tgtcatgcac tggactacat tgttgttgat 1680 tctattgata
tagcccaaga atgtgtaaac ttccttaaaa gacaaaatat tggagttgca 1740
acctttatag gtttagataa gatggctgta tgggcgaaaa agatgaccga aattcaaact
1800 cctgaaaata ctcctcgttt atttgattta gtaaaagtaa aagatgagaa
aattcgccaa 1860 gctttttatt ttgctttacg agatacctta gtagctgaca
acttggatca agccacaaga 1920 gtagcatatc aaaaagatag aagatggaga
gtggtaactt tacagggaca aatcatagaa 1980 cagtcaggta caatgactgg
tggtggaagc aaagtaatga aaggaagaat gggttcctca 2040 cttgttattg
aaatctctga agaagaggta aacaaaatgg aatcacagtt gcaaaacgac 2100
tctaaaaaag caatgcaaat ccaagaacag aaagtacaac ttgaagaaag agtagttaag
2160 ttacggcata gtgaacgaga aatgaggaac acactagaaa aatttactgc
aagcatccag 2220 cgtttaatag agcaagaaga atatttgaat gtccaagtta
aggaacttga agctaatgta 2280 cttgctacag cccctgacaa aaaaaagcag
aaattgctag aagaaaacgt tagtgctttc 2340 aaaacagaat atgatgctgt
ggctgagaaa gctggtaaag tagaagctga ggttaaacgc 2400 ttacacaata
ccatcgtaga aatcaataat cataaactca aggcccaaca agacaaactt 2460
gataaaataa ataagcaatt agatgaatgt gcttctgcta ttactaaagc ccaagtagca
2520 atcaagactg ctgacagaaa ccttcaaaag gcacaagact ctgtcttgcg
tacagagaaa 2580 gaaataaaag atactgagaa agaggtggat gacctaacag
cagagctgaa aagtcttgag 2640 gacaaagcag cagaggtcgt aaagaataca
aatgctgcag aggaatcctt accagagatc 2700 cagaaagaac atcgcaatct
gcttcaagaa ttaaaagtta ttcaagaaaa tgaacatgct 2760 cttcaaaaag
atgcacttag tattaagttg aaacttgaac aaatagatgg tcacattgct 2820
gaacataatt ctaaaataaa atattggcac aaagagattt caaaaatatc actgcatcct
2880 atagaagata atcctattga agagatttcg gttctaagcc cagaggatct
tgaagcgatc 2940 aagaatccag attctataac aaatcaaatt gcacttttgg
aagcccggtg tcatgaaatg 3000 aaaccaaacc tcggtgccat cgcagagtat
aaaaagaagg aagaattgta tttgcaacgg 3060 gtagcagaat tggacaaaat
tacttatgaa agagacagtt ttagacaggc atatgaagat 3120 cttcggaaac
aaaggcttaa tgaatttatg gcaggttttt atataataac aaataaatta 3180
aaggaaaatt accaaatgct tactttggga ggggacgccg aactcgagct tgtagacagc
3240 ttggatcctt tctctgaagg aatcatgttc agtgttcgac cacctaagaa
aagttggaaa 3300 aagatcttca acctttcggg aggagagaaa acacttagtt
cattggcttt agtatttgct 3360 cttcaccact acaagcccac tcccctttac
ttcatggatg agattgatgc agcccttgat 3420 tttaaaaatg tgtccattgt
tgcattttat atatatgaac aaacaaaaaa tgcacagttc 3480 ataataattt
ctcttcgaaa taatatgttt gagatttcgg atagacttat tggaatttac 3540
aagacataca acataacaaa aagtgttgct gtaaatccaa aagaaattgc atctaaggga
3600 ctttgttgaa ctttatctga agtctcaagt tgattcaggt attactgatt
tttttctatt 3660 tgtaaaggat tatgagttgt ataaaataca tactccctaa
actagatcat gaaactggtt 3720 tctgttttat gcagttgtca tttgtaaagt
ctaataaaat attctctata attgcttcta 3780 gattacaaaa atatgacaa 3799 25
429 DNA Homo sapiens misc_feature (1)...(429) n = A,T,C or G 25
atgggaacaa agaagtattt taaaattata actactcatt ctttctttag ccttagttaa
60 tttgagcaga agccacaaca agcaaaccac aataaattta gaattggcag
aaatccacat 120 taactcctct tcccaagttt ccacactact accatttaca
gttgtaggtt tgtaatgtat 180 aattatgtaa tgcagaaact agctttgact
tgtgtaacga tgcactgtca aagtaagcaa 240 agtaagaatt gaaattccac
attcccagaa tttaacactc agctgctcct ctagtaataa 300 gttcctgggg
ataatacatt aaccaacatt ggttgaaaca tacctgagta atcatatcag 360
gatgcatgtt aagctgataa aacaataaga tcccaaaatg cagtagctca aaaaaaaaaa
420 aaaaaaggn 429 26 788 DNA Homo sapiens misc_feature (1)...(788)
n = A,T,C or G 26 nccttttttt tttttttttt gagctactgc attttgggat
cttattgttt tatcagctta 60 acatgcatcc tgatatgatt actcaggtat
gtttcaacca atgttggtta atgtattatc 120 cccaggaact tattactaga
ggagcagctg agtgttaaat tctgggaatg tggaatttca 180 attcttactt
tgcttacttt gacagtgcat cgttacacaa gtcaaagcta gtttctgcat 240
tacataatta tacattacaa acctacaact gtaaatggta gtagtgtgga aacttgggaa
300 gaggagttaa tgtggatttc tgccaattct aaatttattg tggtttgctt
gttgtggctt 360 ctgctcaaat taactaaggc taaagaaaga atgagtagtt
ataattttaa aatacttctt 420 tgttcccata tagcaccctt tacgcgctga
gatgaaaaaa cactttttgt tgagactaag 480 agcttattac tcttcccaag
attctctggc aattcagatt ccccaacttc catatcagcc 540 attttcttct
aataaaggaa ctactgatat tcttgggcaa attattacct cctctggctc 600
agttgttttg accatgggct aatgagccca gggcctgggg tttgattccc acgcatgcca
660 attagctttg cttgcctcca ccaacccagg ctgccctatt aaagcctgcc
gcctgtccga 720 agatgccacc acacatcttg ccttatgagt cattggtcat
aaaaggggcc agctaatgag 780 tagggaaa 788 27 687 DNA Homo sapiens
misc_feature (1)...(687) n = A,T,C or G 27 acatggtttg tgctttactc
ttaaacatct ttaaagtgct attattctat atctgttgga 60 tgagtcatta
tttttgaaat gataatccta gcatgaactc tgatctatgg tgttggattc 120
tgtttcttaa ataactttaa aattaactgt tttcccttga gatttccttc tcctatgtag
180 gtatttgagc tattgttcta agtttacctg taagtataaa ccttgggaga
atctaagtaa 240 acatatttct aaaagcatag ttaccttcct attttctggc
tcttaccttc ttggagtatt 300 taaatgccca tttgccaaaa gcagacctga
acatcaagcc tgttaattct tcaaagaatt 360 taggtatttg tttcaccgaa
atgaagtgac ttattagcca ttcagcgtat tagtattaca 420 gaggctcttg
cccagccaca tccattcatt gatttttatg gctactcttc ccagttacat 480
tttatgcatc tgtaagcttt ccttccttag caaaattgca ttcaaaaatg tgtaaaaatg
540 agtaaataca gaatatcact acagagactt gnatcctcan ggttaatgga
tttcacattg 600 ngaaataaac agcaaanggt cttaagtttt caagtgaaaa
ctttttgggt aatcacaaaa 660 atacctggac acataccacg ctttaaa 687 28 1529
DNA Homo sapiens misc_feature (1)...(1529) n = A,T,C or G 28
gagatcatcg atttaggtgg ctgcntaagt attactgatg tgtccttaca tgcattagga
60 aaaaactrcm cmttwtwgca gtgtgtcgac ttttcagcta ctcaggtatc
tgacagtggt 120 gtgattgcac ttgttagtgg accttgtgcg aagaaattag
aggagattca tatgggacat 180 tgtgtaaatc tgactgatgg ggctgtcgaa
gctgtcctta cttactgtcc tcaaatacgt 240 atattactct tccatggatg
ccccttgata acagatcatt cccgagaagt gttggagcaa 300 ttagtaggcc
caaacaaact aaagcaagtg acatggactg tttattgatg cttttttgaa 360
gatgatcaat gctaggaaag cttatcaaaa ctactttccc aggaaaccat ctatagagat
420 ttgcattcta cttaatgtta acactatttt taattatttt attgtcttaa
gttataactc 480 tcagagaatt agctaagtct tggtatatac atggtttgtg
ctttactctt aaacatcttt 540 aaagtgctat tattctawaw mtgttggatg
agtcattatt tttgaaatga taatcctagc 600 atgaactctg atctatggtg
ttggattctg tttcttaaat aactttaaaa ttaactgttt 660 tcccttgaga
tttccttctc ctatgtaggt atttgagcta ttgttctaag tttacctgta 720
agtataaacc ttgggagaat ctaagtaaac atatttctaa aagcatagtt accttcctat
780 tttctggctc ttaccttctt ggagtattta aatgcccatt tgccaaaagc
agacctgaac 840 atcaagcctg gttaattctt caaagaattt aggkgattkg
tttcmccgga aatgragtga 900 cttattagcc attcagcggt attagkawta
cagaggctct tgcccagcca catccantyc 960 attgattttt awggctactc
ttcccagtta cattttatgc atctgtaagc tttccttcct 1020 tagcaaaatt
gcattcaaaa atgtgtaaaa atgagtaaat acagaatatc actacagaga 1080
cttgtatcct caggtttatt gatttcacat tgtgaaataa acagcaaagg tcttagtttt
1140 caagtgaaaa ctttttggta atcacaaaat tacctgacac ataccacgct
ttaaaccaac 1200 ccccaaattt agcatattca ttttgccatg agccagtctt
gagattttct taaaagattt 1260 cttattttgc ctctgatgta gtgaaaaacg
gggtaagtat gctaactttc ttgtatatgt 1320 tggggggtac ttattcaact
ccatttcttg tccttacaag atttataaat gtggtatgtt 1380 tatagtgtgg
atatatatgt tgccactgca aaggtggtgc atatgtatat atgtgcaaaa 1440
tgggtaaggc ctgttctaac tatgaaattt ttctaaagac aaattcaata aaatttaata
1500 ctgaatattt aamcaagtca aaaaaaaaa 1529 29 697 DNA Homo sapiens
misc_feature (1)...(697) n = A,T,C or G 29 aaaaaagaaa gaaagacaag
aaaaagaaaa aaaaaagaaa cacctttgtc tttgtacacg 60 tcacgngggc
tcccaggaaa atgttccttc tctttttgtt ggcatgggca ctgtgggatc 120
tggngcattc cggtcgacac tctcgtttat ttggactgta agtctgacct ctatgaataa
180 ttacttcagc ccctgattgc tcccgtgcca agctccttgg ccaaactttc
accttagctt 240 ctggtaagtc ttgggccaag ctaagcagca tctatcaatc
atcccttcag ctcctgattg 300 gtcctgggcc aaaggcctgg gccaagctga
gccacacgtt tttcaagaca gcctgtgaac 360 taggcacatt tccttccctt
cccagtcctt aaaaaccctg gacccagcct cgtagagggc 420 accactttca
gacacctatc tctgctggca aagagctttc ttctcttgct tcttaaactt 480
tcactccaac ctcacctttg ngtttacact ccttaatctc cttagaggta gaacaaagaa
540 ctctggatgg tatctcagac tacgagagac tggtacatct tggngcactg
ctgagactat 600 gacacttggg ttctttgagg ttggactaaa tattttacat
ggagggaaat aatacaggct 660 ttcnttttga ctggcntaat ttacttaacn aaaaagg
697 30 1165 DNA Homo sapiens misc_feature (1)...(1165) n = A,T,C or
G 30 aatgctaagt ccaaagtggt taagtgacct gcccaagctc tacaatgccc
tcctgaactc 60 ggatgtcttc atttcctgtg ccagactctt aaaaaaaata
aaaataaata aaaaaagaaa 120 gtacatctaa aaaagaaaga aagacaagaa
aaagaaaaaa aaaagaaaca cctttgtctt 180 tgtacagtca gtgggctccc
aggaaaatgt tccttctctt tttgttggca tgggcactgt 240 gggatctggt
gcattccggt cgacactctc gtttatttgg actgtaagtc tgacctctat 300
gaataattac ttcagcccct gattgctccc gtgccaagct ccttggccaa actttcacct
360 tagcttctgr taagtcttgg gccaagctaa gcagcatcta tcaatcatcc
cttcagctcc 420 tgattgrtcc ygggccaaag gcctgggcca aagctgagcc
acacgttttt caagacagcc 480 tgtgaactag gcacatatcc ttcccttccc
agtccataaa aaccctggac ccagcctcgt 540 agaggcacca ctttcagaca
cctatctctg ctggcaaaga gctttcttct cttgcttctt 600 aaactttcac
tccaacctca cctttgtgtt yacrctcctt aatctcctta gaggtagaac 660
aaagaactct ggatgttatc tcagactacg agagactgtt acatcttggt gcactgctga
720 gactaygaca cttggtttct ttgagtttga ctaaatattt tacatgagtg
taattawtac 780 agctttcctt tttgactgtc ttattttact taacagaatg
ttttgaagga tttgtccyta 840 ttgttagtac ttttcaagat ttccttattt
ttaaggstgr atgctatccc acgtggattg 900 tacgtgccct gtttgctgaa
tctactcatc cttaagggta catttgcttc caggtaacat 960 gtttgtgact
aatactacaa atgtgcatat atctattcca tgttctgctt tggtctgttt 1020
ggggatattt ttccatacac tggattcagt accatggtgg taatcccctt gctnttggtt
1080 gncctcaatc cgggtggatg gnacggtccc ccccaaaatt aattggccca
cggaccaagg 1140 tggtcaanga aggcctcnac cccct 1165 31 557 DNA Homo
sapiens misc_feature (1)...(557) n = A,T,C or G 31 cgcttagggc
cctcgcgggg ggcttgtggg tcctcctccc cctcccactg acaactgccc 60
caactgctct tcccgccccg gtcacagtga aaatgtagac ggggtcgttg tccgtacgac
120 tgtgcgccag ggctcgggga ggggcgccct ccgcgtgagc gcccccctgg
gaatattgaa 180 cataatcacc tctcattcca gactatgtta ggtcttaatg
gtgggaggac gcccgagtgc 240 tcggcccgtt tcaccccgag gaggaaggac
actgggtcat gacgccatca gagggcgcca 300 gagcagggac cggacgcgag
ttggagatgt tggactcgct gttggccttg ggcggctggt 360 gctgcttcgg
gattccgtgg agtgggaggg gcgcagtctc ttgaaggcgc ctgtccaaga 420
aagagagaga agccagagat agcctgatcc tgccttncag ttcagttctg aaaaacagca
480 ggctcttctg cggnctaggc canggcaggc taccagccac atcttctatg
agccagatgc 540 ttatgatgac ctggacc 557 32 527 DNA Homo sapiens
misc_feature (1)...(527) n = A,T,C or G 32 atccagggag aggagtctat
ctcctcaagn ttgacaactc ctactctttg tggcggncaa 60 aatcagtcta
ctacagagtc tattatacta gataaaaatg tnggtacaaa gtctggagtc 120
tagggttggg cagaagatga catttaattt ggaaatttct ttttactttt gtggagcatt
180 agagtcacag tttaccttat tgatattggt ctgatggntt gtgaactctt
gctgggaatc 240 aaaatttcct tgagactctt tagcattcat actttggggn
taaaggagat tnctcagact 300 catccagccc ttgggtgctg accagcagag
tcactagngg atgctgaagt tacatgagct 360 acatgttaaa tatttaaagt
ctccaaaata aaacacccca acgttgacct tacccggctt 420 gatggttagc
ccctttgctg gctgctccat gtgccttatg agagcccgta agttacaggt 480
gtcctctaat ttgaaatcca taagntaaca ngtctatatc agntgcn 527 33 934 DNA
Homo sapiens 33 gtaggccagc gatgacgacg aggaggaaga aggaaacatc
ggttgtgaag agaaagccaa 60 aaagaatgcc aacaagcctt tgctggatga
gattgtgcct gtgtccgacg ggactgtcat 120 gaggatgtgt atgctggcag
ccatcaatat ccaagggaga ggagtctatc tcctcaagtt 180 tgacaactcc
tactctttgt ggcggtcaaa atcagtctac tacrgagtct attatactag 240
ataaaaatgt tgttacaaag tctggagtct wgggttgggc agaagatgac atttaatttg
300 gaaatttctt tttacttttg tggagcatta gagtcacagt ttaccttatt
gatattggtc 360 tgatggtttg tgaactcttg ctgggaatca aaatttcctt
gagactcttt agcattcata 420 ctttggggtt aaaggagatt cctcagactc
atccagccct tgggtgctga ccagcagagt 480 cactagtgga tgctgaagtt
acatgagcta catgttaaat atttaaagtc tccaaaataa 540 aacaccccaa
cgttgacctt acccggctga tggttagccc cttgctgcct gctccatgtg 600
tcttatgaga gcccgtagtt acagtgtcct ctaatttgaa atccataagt taacaagtct
660 atatcaggtg cagctggctt tgattaaagg ccatttttaa aacttaaaaa
ctcaacacct 720 cacagattat aatagaaaaa mgaaatgggc ctcagtttga
tctccgttca gaatgaccca 780 gattgtttct gctttggggt gcagctgttt
aagttcagag ttatattaca gagaattatt 840 ttyctggaga taatctttaa
acctagaatg kttcaaaacc waattggata attggaagta 900 tccaagatac
gtagaacacc cccggagaat tttc 934 34 758 DNA Homo sapiens misc_feature
(1)...(758) n = A,T,C or G 34 ggctttatag cccatcctca ttgcttactg
ccacccctca gctggggtcc aaggcagtac 60 tattcagttt attcaccaga
cctgcctcca gacatctact tctttcaaaa attagtgttt 120 tccatcaagg
agcatgttcc agagcatttc ccagagatgt cccaaagaac actgtccggt 180
gctgtggcgt acagtggcaa cagcattaga ctaagtggaa catcccagca ggctgcttta
240 gaatccgctc atttgactag atacgatgta attggctgtc tttaaaaaac
gcgcacacac 300 acacaatctg ataggcatat ctcatgccca ttcaatatgg
aatgttcttc gcttgctgaa 360 tttaagcctg tattttaagg ttttgtggtt
cctcggccac aatgggtgat gtcactgata 420 gaacgaagct gagtttccaa
gggtttgggg ctgtgcaaga gtaaacacta gagcttgagt 480 tgttatccag
ctggcaagca cggaagtctt tgaagaatgt aatgtaaaaa gggaaaagaa 540
tgtaaagctt tttgtaccaa atgagagttg gagcccagcc aacaaatgct tttccctgtg
600 taaaagtctc tctggaaggg acattccatc tccatggtgc actctgaggg
gcactgtcaa 660 ctagagattg gccccatcca ggtgggagga acccctttgg
gatggngagt atncaatctg 720 ctgngcattt tgacaggatc tctgaatggc taggtaat
758 35 1534 DNA Homo sapiens misc_feature (1)...(1534) n = A,T,C or
G 35 ngaggtaaaa ggcaaggcag catttaataa gtacctgttg tatcctttta
agtgtttgtt 60 gtggtaatcc tcacaaagac cgggactgat ggaaactcct
tgctattaaa ctttttttct 120 tgaggaattt tgcttttcaa gtgcatatac
actattaata ttttttaccc aagaggagca 180 ttctaagcta atttatgcag
tgtgactgta ttaagcatta agcttccttc agagctggcc 240 tatcggagat
gctactgccc tctctacaga tgtgtctgaa atgcctgccc aaggatggcc 300
cttagccagt taacagcttt atagcccatc ctcattgctt actgccaccc ctcagctggg
360 gtccaaggca gtactattca gtttattcac cagacctgcc tccagacatc
tacttctttc 420 aaaaattagt gttttccatc aaggagcatg ttccagagca
tttcccagag atgtcccaaa 480 gaacactgtc cggtgctgtg gcgtacagtg
gcaacagcat tagactaagt ggaacatccc 540 agcaggctgc tttagaatcc
gctcatttga ctagatacga tgtaattggc tgtctttaaa 600 aaacgcggca
cacacacaca atctgatagg gcatatctca tgcccattca atatggaatg 660
ttcttcgctt gctgaattta agcctgtatt ttaaggtttt gtggttcctc ggccacaatg
720 gggtgatgtc actgatagaa cgaagctgag tttccaaggg tttggggctg
tgcaaggagt 780 aaacactaga gcttgagttg ttatccagct ggcaagcacg
gaagtctttg aagaatgtaa 840 tgtaaaaagg gaaaagaatg taaagctttt
tgtaccaaat gagagttgga gcccagccaa 900 caaatgcttt tccctgtgta
aaagtctctc tggaagggac attccatctc catggtgcac 960 tctgaggggc
actgtcaact agagattggc cccatccagg tgggaggaac
ccctttggrr 1020 tggtgagtat ccaatctgct gtgcatttga caggatctct
gaatggctag gtaatggatc 1080 ccaagcaggc tcacaaattt aaatgagggc
tttgtgtgca gaaagaggaa taagtacaga 1140 ttattttcct accactagat
ttttggggag agtcaccatg gaatgttgac aattacttaa 1200 aatattttaa
gctcccttgc tgaattcctg tcctgtccct gaggaatcag atggtcatac 1260
agccataggc acccacccga aatttcccta ggagttggag taatgctaga attgaagacc
1320 ttctgagtaa agggcttctc tgccttctca gaggcaggag aattttgcac
tggttgtgtt 1380 aaatgtataa aaagctatat gttcaccagt ttactcattt
ccaatgtgta gatgaataaa 1440 atgtagtgta caaattattt gaaaatccca
gaaggaaggt acttttcaaa tacagtattt 1500 tttttaacaa ataaacttac
gatttttaca gcaa 1534 36 125 PRT Homo sapiens variant (1)...(125)
Xaa = Any amino acid 36 Leu Ser Ser Arg Gly Met Lys Ala Val Leu Leu
Ala Asp Thr Glu Ile 5 10 15 Asp Leu Phe Ser Thr Asp Ile Pro Pro Thr
Asn Ala Val Asp Phe Thr 20 25 30 Gly Arg Cys Tyr Phe Thr Lys Ile
Cys Lys Cys Lys Leu Lys Asp Ile 35 40 45 Ala Cys Leu Lys Cys Gly
Asn Ile Val Xaa Tyr His Val Ile Val Pro 50 55 60 Cys Ser Ser Cys
Leu Leu Ser Cys Asn Asn Arg His Phe Trp Met Phe 65 70 75 80 His Ser
Gln Ala Val Tyr Asp Ile Asn Arg Leu Asp Ser Thr Gly Val 85 90 95
Asn Val Leu Leu Arg Gly Asn Leu Pro Glu Ile Glu Glu Ser Thr Asp 100
105 110 Glu Asp Val Leu Asn Ile Ser Ala Glu Glu Cys Ile Arg 115 120
125 37 448 PRT Homo sapiens VARIANT (1)...(448) Xaa = any amino
acid 37 Met Ser Arg Arg Pro Cys Ser Cys Ala Leu Arg Pro Pro Arg Cys
Ser 5 10 15 Cys Ser Ala Ser Pro Ser Ala Val Thr Ala Ala Gly Arg Pro
Arg Pro 20 25 30 Ser Asp Ser Cys Lys Glu Glu Ser Ser Thr Leu Ser
Val Lys Met Lys 35 40 45 Cys Asp Phe Asn Cys Asn His Val His Ser
Gly Leu Lys Leu Val Lys 50 55 60 Pro Asp Asp Ile Gly Arg Leu Val
Ser Tyr Thr Pro Ala Tyr Leu Glu 65 70 75 80 Gly Ser Cys Lys Asp Cys
Ile Lys Asp Tyr Glu Arg Leu Ser Cys Ile 85 90 95 Gly Ser Pro Ile
Val Ser Pro Arg Ile Val Gln Leu Glu Thr Glu Ser 100 105 110 Lys Arg
Leu His Asn Lys Glu Asn Gln His Val Gln Gln Thr Leu Asn 115 120 125
Ser Thr Asn Glu Ile Glu Ala Leu Glu Thr Ser Arg Leu Tyr Glu Asp 130
135 140 Ser Gly Tyr Ser Ser Phe Ser Leu Gln Ser Gly Leu Ser Glu His
Glu 145 150 155 160 Glu Gly Ser Leu Leu Glu Glu Asn Phe Gly Asp Ser
Leu Gln Ser Cys 165 170 175 Leu Leu Gln Ile Gln Ser Pro Asp Gln Tyr
Pro Asn Lys Asn Leu Leu 180 185 190 Pro Val Leu His Phe Glu Lys Val
Val Cys Ser Thr Leu Lys Lys Asn 195 200 205 Ala Lys Arg Asn Pro Lys
Val Asp Arg Glu Met Leu Lys Glu Ile Ile 210 215 220 Ala Arg Gly Asn
Phe Arg Leu Gln Asn Ile Ile Gly Arg Lys Met Gly 225 230 235 240 Leu
Glu Cys Val Asp Ile Leu Ser Glu Leu Phe Arg Arg Gly Leu Arg 245 250
255 His Val Leu Ala Thr Ile Leu Ala Gln Leu Ser Asp Met Asp Leu Ile
260 265 270 Asn Val Ser Lys Val Ser Thr Thr Trp Lys Lys Ile Leu Glu
Asp Asp 275 280 285 Lys Gly Ala Phe Gln Leu Tyr Ser Lys Ala Ile Gln
Arg Val Thr Glu 290 295 300 Asn Asn Asn Lys Phe Ser Pro His Ala Ser
Thr Arg Glu Tyr Val Met 305 310 315 320 Phe Arg Thr Pro Leu Ala Ser
Val Gln Lys Ser Ala Ala Gln Thr Ser 325 330 335 Leu Lys Lys Asp Ala
Gln Thr Lys Leu Ser Asn Gln Gly Asp Gln Lys 340 345 350 Gly Ser Thr
Tyr Ser Arg His Asn Glu Phe Ser Glu Val Ala Lys Thr 355 360 365 Leu
Lys Lys Asn Glu Ser Leu Lys Ala Cys Ile Arg Cys Asn Ser Pro 370 375
380 Ala Lys Tyr Asp Cys Tyr Leu Gln Arg Ala Thr Cys Lys Arg Glu Gly
385 390 395 400 Cys Gly Phe Asp Tyr Cys Thr Lys Cys Leu Cys Asn Tyr
His Thr Thr 405 410 415 Lys Asp Cys Ser Asp Gly Lys Leu Leu Lys Ala
Ser Cys Lys Ile Gly 420 425 430 Pro Leu Pro Gly Thr Lys Lys Ser Lys
Lys Asn Leu Arg Arg Leu Xaa 435 440 445 38 1050 PRT Homo sapiens 38
Met Ala Ala Val Lys Lys Glu Gly Gly Ala Leu Ser Glu Ala Met Ser 5
10 15 Leu Glu Gly Asp Glu Trp Glu Leu Ser Lys Glu Asn Val Gln Pro
Leu 20 25 30 Arg Gln Gly Arg Ile Met Ser Thr Leu Gln Gly Ala Leu
Ala Gln Glu 35 40 45 Ser Ala Cys Asn Asn Thr Leu Gln Gln Gln Lys
Arg Ala Phe Glu Tyr 50 55 60 Glu Ile Arg Phe Tyr Thr Gly Asn Asp
Pro Leu Asp Val Trp Asp Arg 65 70 75 80 Tyr Ile Ser Trp Thr Glu Gln
Asn Tyr Pro Gln Gly Gly Lys Glu Ser 85 90 95 Asn Met Ser Thr Leu
Leu Glu Arg Ala Val Glu Ala Leu Gln Gly Glu 100 105 110 Lys Arg Tyr
Tyr Ser Asp Pro Arg Phe Leu Asn Leu Trp Leu Lys Leu 115 120 125 Gly
Arg Leu Cys Asn Glu Pro Leu Asp Met Tyr Ser Tyr Leu His Asn 130 135
140 Gln Gly Ile Gly Val Ser Leu Ala Gln Phe Tyr Ile Ser Trp Ala Glu
145 150 155 160 Glu Tyr Glu Ala Arg Glu Asn Phe Arg Lys Ala Asp Ala
Ile Phe Gln 165 170 175 Glu Gly Ile Gln Gln Lys Ala Glu Pro Leu Glu
Arg Leu Gln Ser Gln 180 185 190 His Arg Gln Phe Gln Ala Arg Val Ser
Arg Gln Thr Leu Leu Ala Leu 195 200 205 Glu Lys Glu Glu Glu Glu Glu
Val Phe Glu Ser Ser Val Pro Gln Arg 210 215 220 Ser Thr Leu Ala Glu
Leu Lys Ser Lys Gly Lys Lys Thr Ala Arg Ala 225 230 235 240 Pro Ile
Ile Arg Val Gly Gly Ala Leu Lys Ala Pro Ser Gln Asn Arg 245 250 255
Gly Leu Gln Asn Pro Phe Pro Gln Gln Met Gln Asn Asn Ser Arg Ile 260
265 270 Thr Val Phe Asp Glu Asn Ala Asp Glu Ala Ser Thr Ala Glu Leu
Ser 275 280 285 Lys Pro Thr Val Gln Pro Trp Ile Ala Pro Pro Met Pro
Arg Ala Lys 290 295 300 Glu Asn Glu Leu Gln Ala Gly Pro Trp Asn Thr
Gly Arg Ser Leu Glu 305 310 315 320 His Arg Pro Arg Gly Asn Thr Ala
Ser Leu Ile Ala Val Pro Ala Val 325 330 335 Leu Pro Ser Phe Thr Pro
Tyr Val Glu Glu Thr Ala Gln Gln Pro Val 340 345 350 Met Thr Pro Cys
Lys Ile Glu Pro Ser Ile Asn His Ile Leu Ser Thr 355 360 365 Arg Lys
Pro Gly Lys Glu Glu Gly Asp Pro Leu Gln Arg Val Gln Ser 370 375 380
His Gln Gln Ala Ser Glu Glu Lys Lys Glu Lys Met Met Tyr Cys Lys 385
390 395 400 Glu Lys Ile Tyr Ala Gly Val Gly Glu Phe Ser Phe Glu Glu
Ile Arg 405 410 415 Ala Glu Val Phe Arg Lys Lys Leu Lys Glu Gln Arg
Glu Ala Glu Leu 420 425 430 Leu Thr Ser Ala Glu Lys Arg Ala Glu Met
Gln Lys Gln Ile Glu Glu 435 440 445 Met Glu Lys Lys Leu Lys Glu Ile
Gln Thr Thr Gln Gln Glu Arg Thr 450 455 460 Gly Asp Gln Gln Glu Glu
Thr Met Pro Thr Lys Glu Thr Thr Lys Leu 465 470 475 480 Gln Ile Ala
Ser Glu Ser Gln Lys Ile Pro Gly Met Thr Leu Ser Ser 485 490 495 Ser
Val Cys Gln Val Asn Cys Cys Ala Arg Glu Thr Ser Leu Ala Glu 500 505
510 Asn Ile Trp Gln Glu Gln Pro His Ser Lys Gly Pro Ser Val Pro Phe
515 520 525 Ser Ile Phe Asp Glu Phe Leu Leu Ser Glu Lys Lys Asn Lys
Ser Pro 530 535 540 Pro Ala Asp Pro Pro Arg Val Leu Ala Gln Arg Arg
Pro Leu Ala Val 545 550 555 560 Leu Lys Thr Ser Glu Ser Ile Thr Ser
Asn Glu Asp Val Ser Pro Asp 565 570 575 Val Cys Asp Glu Phe Thr Gly
Ile Glu Pro Leu Ser Glu Asp Ala Ile 580 585 590 Ile Thr Gly Phe Arg
Asn Val Thr Ile Cys Pro Asn Pro Glu Asp Thr 595 600 605 Cys Asp Phe
Ala Arg Ala Ala Arg Phe Val Ser Thr Pro Phe His Glu 610 615 620 Ile
Met Ser Leu Lys Asp Leu Pro Ser Asp Pro Glu Arg Leu Leu Pro 625 630
635 640 Glu Glu Asp Leu Asp Val Lys Thr Ser Glu Asp Gln Gln Thr Ala
Cys 645 650 655 Gly Thr Ile Tyr Ser Gln Thr Leu Ser Ile Lys Lys Leu
Ser Pro Ile 660 665 670 Ile Glu Asp Ser Arg Glu Ala Thr His Ser Ser
Gly Phe Ser Gly Ser 675 680 685 Ser Ala Ser Val Ala Ser Thr Ser Ser
Ile Lys Cys Leu Gln Ile Pro 690 695 700 Glu Lys Leu Glu Leu Thr Asn
Glu Thr Ser Glu Asn Pro Thr Gln Ser 705 710 715 720 Pro Trp Cys Ser
Gln Tyr Arg Arg Gln Leu Leu Lys Ser Leu Pro Glu 725 730 735 Leu Ser
Ala Ser Ala Glu Leu Cys Ile Glu Asp Arg Pro Met Pro Lys 740 745 750
Leu Glu Ile Glu Lys Glu Ile Glu Leu Gly Asn Glu Asp Tyr Cys Ile 755
760 765 Lys Arg Glu Tyr Leu Ile Cys Glu Asp Tyr Lys Leu Phe Trp Val
Ala 770 775 780 Pro Arg Asn Phe Ala Glu Leu Thr Val Ile Lys Val Ser
Ser Gln Pro 785 790 795 800 Val Pro Trp Asp Phe Tyr Ile Asn Leu Lys
Leu Lys Glu Arg Leu Asn 805 810 815 Glu Asp Phe Asp His Phe Cys Ser
Cys Tyr Gln Tyr Gln Asp Gly Cys 820 825 830 Ile Val Trp His Gln Tyr
Ile Asn Cys Phe Thr Leu Gln Asp Leu Leu 835 840 845 Gln His Ser Glu
Tyr Ile Thr His Glu Ile Thr Val Leu Ile Ile Tyr 850 855 860 Asn Leu
Leu Thr Ile Val Glu Met Leu His Lys Ala Glu Ile Val His 865 870 875
880 Gly Asp Leu Ser Pro Arg Cys Leu Ile Leu Arg Asn Arg Ile His Asp
885 890 895 Pro Tyr Asp Cys Asn Lys Asn Asn Gln Ala Leu Lys Ile Val
Asp Phe 900 905 910 Ser Tyr Ser Val Asp Leu Arg Val Gln Leu Asp Val
Phe Thr Leu Ser 915 920 925 Gly Phe Arg Thr Val Gln Ile Leu Glu Gly
Gln Lys Ile Leu Ala Asn 930 935 940 Cys Ser Ser Pro Tyr Gln Val Asp
Leu Phe Gly Ile Ala Asp Leu Ala 945 950 955 960 His Leu Leu Leu Phe
Lys Glu His Leu Gln Val Phe Trp Asp Gly Ser 965 970 975 Phe Trp Lys
Leu Ser Gln Asn Ile Ser Glu Leu Lys Asp Gly Glu Leu 980 985 990 Trp
Asn Lys Phe Phe Val Arg Ile Leu Asn Ala Asn Asp Glu Ala Thr 995
1000 1005 Val Ser Val Leu Gly Glu Leu Ala Ala Glu Met Asn Gly Val
Phe Asp 1010 1015 1020 Thr Thr Phe Gln Ser His Leu Asn Lys Ala Leu
Trp Lys Val Gly Lys 1025 1030 1035 1040 Leu Thr Ser Pro Gly Ala Leu
Leu Phe Gln 1045 1050 39 258 PRT Homo sapiens 39 Gly Lys Leu Thr
Gly Ile Ser Asp Pro Val Thr Val Lys Thr Ser Gly 5 10 15 Ser Arg Phe
Gly Ser Trp Met Thr Asp Pro Leu Ala Pro Glu Gly Asp 20 25 30 Asn
Arg Val Trp Tyr Met Asp Gly Tyr His Asn Asn Arg Phe Val Arg 35 40
45 Glu Tyr Lys Ser Met Val Asp Phe Met Asn Thr Asp Asn Phe Thr Ser
50 55 60 His Arg Leu Pro His Pro Trp Ser Gly Thr Gly Gln Val Val
Tyr Asn 65 70 75 80 Gly Ser Ile Tyr Phe Asn Lys Phe Gln Ser His Ile
Ile Ile Arg Phe 85 90 95 Asp Leu Lys Thr Glu Thr Ile Leu Lys Thr
Arg Ser Leu Asp Tyr Ala 100 105 110 Gly Tyr Asn Asn Met Tyr His Tyr
Ala Trp Gly Gly His Ser Asp Ile 115 120 125 Asp Leu Met Val Asp Glu
Ser Gly Leu Trp Ala Val Tyr Ala Thr Asn 130 135 140 Gln Asn Ala Gly
Asn Ile Val Val Ser Arg Leu Asp Pro Val Ser Leu 145 150 155 160 Gln
Thr Leu Gln Thr Trp Asn Thr Ser Tyr Pro Lys Arg Ser Ala Gly 165 170
175 Glu Ala Phe Ile Ile Cys Gly Thr Leu Tyr Val Thr Asn Gly Tyr Ser
180 185 190 Gly Gly Thr Lys Val His Tyr Ala Tyr Gln Thr Asn Ala Ser
Thr Tyr 195 200 205 Glu Tyr Ile Asp Ile Pro Phe Gln Asn Lys Tyr Ser
His Ile Ser Met 210 215 220 Leu Asp Tyr Asn Pro Lys Asp Arg Ala Leu
Tyr Ala Trp Asn Asn Gly 225 230 235 240 His Gln Ile Leu Tyr Asn Val
Thr Leu Phe His Val Ile Arg Ser Asp 245 250 255 Glu Leu 40 324 PRT
Homo sapiens 40 Met Asp Ala Pro Arg Gln Val Val Asn Phe Gly Pro Gly
Pro Ala Lys 5 10 15 Leu Pro His Ser Val Leu Leu Glu Ile Gln Lys Glu
Leu Leu Asp Tyr 20 25 30 Lys Gly Val Gly Ile Ser Val Leu Glu Met
Ser His Arg Ser Ser Asp 35 40 45 Phe Ala Lys Ile Ile Asn Asn Thr
Glu Asn Leu Val Arg Glu Leu Leu 50 55 60 Ala Val Pro Asp Asn Tyr
Lys Val Ile Phe Leu Gln Gly Gly Gly Cys 65 70 75 80 Gly Gln Phe Ser
Ala Val Pro Leu Asn Leu Ile Gly Leu Lys Ala Gly 85 90 95 Arg Cys
Ala Asp Tyr Val Val Thr Gly Ala Trp Ser Ala Lys Ala Ala 100 105 110
Glu Glu Ala Lys Lys Phe Gly Thr Ile Asn Ile Val His Pro Lys Leu 115
120 125 Gly Ser Tyr Thr Lys Ile Pro Asp Pro Ser Thr Trp Asn Leu Asn
Pro 130 135 140 Asp Ala Ser Tyr Val Tyr Tyr Cys Ala Asn Glu Thr Val
His Gly Val 145 150 155 160 Glu Phe Asp Phe Ile Pro Asp Val Lys Gly
Ala Val Leu Val Cys Asp 165 170 175 Met Ser Ser Asn Phe Leu Ser Lys
Pro Val Asp Val Ser Lys Phe Gly 180 185 190 Val Ile Phe Ala Gly Ala
Gln Lys Asn Val Gly Ser Ala Gly Val Thr 195 200 205 Val Val Ile Val
Arg Asp Asp Leu Leu Gly Phe Ala Leu Arg Glu Cys 210 215 220 Pro Ser
Val Leu Glu Tyr Lys Val Gln Ala Gly Asn Ser Ser Leu Tyr 225 230 235
240 Asn Thr Pro Pro Cys Phe Ser Ile Tyr Val Met Gly Leu Val Leu Glu
245 250 255 Trp Ile Lys Asn Asn Gly Gly Ala Ala Ala Met Glu Lys Leu
Ser Ser 260 265 270 Ile Lys Ser Gln Thr Ile Tyr Glu Ile Ile Asp Asn
Ser Gln Gly Phe 275 280 285 Tyr Val Ser Val Gly Gly Ile Arg Ala Ser
Leu Tyr Asn Ala Val Thr 290 295 300 Ile Glu Asp Val Gln Lys Leu Ala
Ala Phe Met Lys Lys Phe Leu Glu 305 310 315 320 Met His Gln Leu 41
410 PRT Homo sapiens 41 Met Glu Ala Glu Asn Ala Gly Ser Tyr Ser Leu
Gln Gln Ala Gln Ala 5 10 15 Phe Tyr Thr Phe Pro Phe Gln Gln Leu Met
Ala Glu Ala Pro Asn Met 20 25 30 Ala Val Val Asn Glu Gln Gln Met
Pro Glu Glu Val Pro Ala Pro Ala 35 40 45 Pro Ala Gln Glu Pro Val
Gln Glu Ala Pro Lys Gly Arg Lys Arg Lys 50 55 60 Pro Arg Thr Thr
Glu Pro Lys Gln Pro Val Glu Pro Lys Lys Pro Val 65 70 75 80 Glu Ser
Lys Lys Ser Gly Lys Ser Ala Lys Pro Lys Glu Lys Gln Glu 85 90 95
Lys Ile Thr Asp Thr Phe Lys Val Lys Arg Lys Val Asp Arg Phe Asn 100
105 110 Gly Val Ser Glu Ala Glu Leu Leu Thr Lys Thr Leu
Pro Asp Ile Leu 115 120 125 Thr Phe Asn Leu Asp Ile Val Ile Ile Gly
Ile Asn Pro Gly Leu Met 130 135 140 Ala Ala Tyr Lys Gly His His Tyr
Pro Gly Pro Gly Asn His Phe Trp 145 150 155 160 Lys Cys Leu Phe Met
Ser Gly Leu Ser Glu Val Gln Leu Asn His Met 165 170 175 Asp Asp His
Thr Leu Pro Gly Lys Tyr Gly Ile Gly Phe Thr Asn Met 180 185 190 Val
Glu Arg Thr Thr Pro Gly Ser Lys Asp Leu Ser Ser Lys Glu Phe 195 200
205 Arg Glu Gly Gly Arg Ile Leu Val Gln Lys Leu Gln Lys Tyr Gln Pro
210 215 220 Arg Ile Ala Val Phe Asn Gly Lys Cys Ile Tyr Glu Ile Phe
Ser Lys 225 230 235 240 Glu Val Phe Gly Val Lys Val Lys Asn Leu Glu
Phe Gly Leu Gln Pro 245 250 255 His Lys Ile Pro Asp Thr Glu Thr Leu
Cys Tyr Val Met Pro Ser Ser 260 265 270 Ser Ala Arg Cys Ala Gln Phe
Pro Arg Ala Gln Asp Lys Val His Tyr 275 280 285 Tyr Ile Lys Leu Lys
Asp Leu Arg Asp Gln Leu Lys Gly Ile Glu Arg 290 295 300 Asn Met Asp
Val Gln Glu Val Gln Tyr Thr Phe Asp Leu Gln Leu Ala 305 310 315 320
Gln Glu Asp Ala Lys Lys Met Ala Val Lys Glu Glu Lys Tyr Asp Pro 325
330 335 Gly Tyr Glu Ala Ala Tyr Gly Gly Ala Tyr Gly Glu Asn Pro Cys
Ser 340 345 350 Ser Glu Pro Cys Gly Phe Ser Ser Asn Gly Leu Ile Glu
Ser Val Glu 355 360 365 Leu Arg Gly Glu Ser Ala Phe Ser Gly Ile Pro
Asn Gly Gln Trp Met 370 375 380 Thr Gln Ser Phe Thr Asp Gln Ile Pro
Ser Phe Ser Asn His Cys Gly 385 390 395 400 Thr Gln Glu Gln Glu Glu
Glu Ser His Ala 405 410 42 484 DNA Homo sapiens 42 ttcacgtaag
actttttggt ttgatcatct ttgttgaggt aggactatca gttccctcta 60
aatgtatatg ttgatttatg agtaattgtt atttattctt tatttattta tattaattat
120 gaagattatg atattatttg attgcagatt tttttggcgc gctgccccct
ccccaccctg 180 ccactcttga cattccactg tgcgttttag aagagagcct
ttttctaaag ggatctgctt 240 aaagttttaa cttttatacc tatctgagtg
aattacagac aacctatcat ttattctgct 300 tcgagggtcc ccagggccct
tgtacaaccg acagctctta cttttaaatg caatctcttt 360 tctacataca
ttattttctt aattgttagc tatttataga aagcttcaat agaactgttt 420
caactgtata actatttact attcaaataa aatattttca aagtcaaaaa aaaaaaaaaa
480 aaag 484 43 700 DNA Homo sapiens 43 ctcaccagta attccactcc
catgaaactt tggtcattgt tatgcattaa gtggggctta 60 tctttggttt
ggagttcatt tgaactcttg aaccttagtt tagtgaagat gaactgtctg 120
ttcttaggta gaaacggtgt ttatttaaaa atcagtttta aaaaatgagc taccatatgt
180 gctgtctatt ataaatggga caccaaacaa aattttctat tacagttgtg
tacttgcaaa 240 cattttgcta tacagtactt catagatgca tacaaatgag
ctcacttatt acaaagacaa 300 acgtttaatt tgctaaatat tttaacaagt
ttgttatata ttttatttaa tttaaaagaa 360 atctcttacc aacctacata
tttattacta taatttgcta tgacttcagg ttaatttatt 420 tgtgtttgca
tagtttgagc aggatgtttt gtgaagtatg tttgtattta tttgcctact 480
ttgtacttga tgtgttttgt aatgtgcact gaatttgttt tcttttcaac tatgttaatg
540 atcaatactg taaattgggt cttttgtaaa caaaaaggca atgatgtatg
catttttttt 600 aatttgaggt agtttgtttg tatactgttt ctccaaacac
ttaatatttc ttacatcaaa 660 gcaacaaaat tgtgttcagt gctgtacatt
tggtgtatgg 700 44 672 DNA Homo sapiens misc_feature (1)...(672) n =
A,T,C or G 44 tttttgttta cataattgta aggaacagta attctagaaa
cactagaaga aaaargcata 60 gcaatgtcca cagttaaaaa aaaaagkgca
cattactcgg tcacaatcac agtcattact 120 tgaaaaacta tatgtaacaa
gtagataaga aatatcactg atgcctcaaa ctcattgtca 180 aaaactgaat
gacataaatt ttacatgaaa taaggcaaat tcaggaatgc acaaagaatt 240
tgtaatccaa ccaaatctaa acaacagaaa aaagttgtat aagaagcatg aactaaagta
300 cttctcccta aatatttaaa aaataggctt gtctcagtgc acaaagaaaa
catcactcat 360 gtgtatccca cactataaaa taagaaagaa gggtaaagta
tgggggatag gagggcacag 420 ttcattgtaa gttgcagctg catccgctga
gagttcctta cattattttt agctagaact 480 gaaaattata caaatcatat
caggagatgt aatggtcttt ttggaaacta tttctgaaag 540 aaatgaaaag
aaaactacac acaagagtgc aaattttcag attgtcactt gcaacctctt 600
aacattcagt catctacatc caggtgctgc tagagggatg cctggagaca gcagcggcaa
660 tcaggaacga gc 672 45 480 DNA Homo sapiens 45 tcagttccat
gtatacaatt accagatgcc accgcagtgc cctgttgggg agcaaaggag 60
aaatctgtgg accgaagcat acaaatggtg gtatcttgtc tgtttaatcc agagaagaga
120 ctgataaatt ccgttgttac tcaagatgac tgcttcaagg gtaaaagagt
gcatcgcttt 180 agaagaagtt tggcagtatt taaatctgtt ggatcctctc
agctatctag tttcatggga 240 agttgctggt tttgaatatt aagctaaaag
ttttccacta ttacagaaat tctgaatttt 300 ggtaaatcac actgaaactt
tctgtataac ttgtattatt agactctcta gttttatctt 360 aacactgaaa
ctgttcttca ttagatgttt atttagaacc tggttctgtg tttaatatat 420
agtttaaagt aacaaataat cgagactgaa agaatgttaa gatttatctg caaggatttt
480 46 427 DNA Homo sapiens misc_feature (1)...(427) n = A,T,C or G
46 tttttaaaaa taagtgtcct actattgtat tatatattga tacgaaactg
ttaaagctat 60 tttgaaaata tgagttctta gctttaatca tgaagtctga
agtttgcttt cagtaattat 120 tttaaaagtt gttttggttc attgctttat
aatatttatt attgaatgcc aaacctgttc 180 ttttttttac tgtgtccaat
attctttcaa gcaaatgcaa tggctggaat ataattcaga 240 attaactgaa
acccagccag aagagggacc acctgtaaag caagtccttt caagtttcac 300
tgcacatccc aaaccatgtt acaaaaagag caactgctat attcacatta tgatattttt
360 ctatcttaaa tttgtcaaaa taaagtatga gtctaactat taaaaaaaaa
aaaaccctck 420 tsccaaa 427 47 581 DNA Homo sapiens 47 tcttttgaaa
aataaaggat ctaatgtctc cctaataagt cttctttcct tccaactaaa 60
tgacctacac ggacttttat tttcttgatc aaagaggtgt ttattaagga cttctggata
120 actatacttt tactctattt ttaaagatca caaagtaatt ttaaatgtga
acaggttccc 180 ataccatgaa tgctggcctc accttctcta tcatccacat
tttgaaatgc aaagaaagct 240 cccttgtaag ccatacttcc ttccccactc
ccatcctagg atacttgccc agtgctcatt 300 aggcatttct tattcagata
gtccaaattt aggttattat gcttaatttg acacattaac 360 taaatgccca
gttttaaaat atatccatca attcacgctg aaatgtgctt ctttgtgcta 420
tcaaatggaa tagaatacac ttatttttta aacaatccca gaatactgtg tgtagacttt
480 tgttgtgctc aaataaatgt ttacttatct tacaaagctc aaatactgga
ttgtaaccat 540 gtgatgaagt tatctatgtt gtacctaaca tgcaaattat c 581 48
491 DNA Homo sapiens misc_feature (1)...(491) n = A,T,C or G 48
ccgggccccc cctcgagggy ttcaatggtc agatggaaca gttgaaaggc gcggtcgaaa
60 ccctcgccat cacgatcgcg caatctggca ttctggaatt cgtcacaacg
atcgtcaccg 120 ccttgggcaa ctttgtcgat aagctcgccg aggtcagccc
ggaaactctg aagtgggtca 180 cgatcatcgg tggggtggcg gcggtgctag
gtccggtggc gatcggcatc ggcgccgtgg 240 tctctgcgct gggcgccttt
ctccctgtca tcgtgcctgt tgcgagcgcc atcggcgctg 300 tcgtttcggt
catcacggcc ggtgccatcc cagccctggc cgggcttgtt gttgccctat 360
cgcctgtgct cgtgccgctg gcggcggtgg ctgctgcagt cggcgccgtt tatctggtgt
420 ggaagaactg ggacatgatc gggcccattc tcgccaagct ttataacgga
gtgaagacgt 480 ggctggtcga t 491 49 1929 DNA Homo sapiens
misc_feature (1)...(1929) n = A,T,C or G 49 ttaggctagt agaggctggt
gttaatcggc cgagggccgc tgtcaggttg gagtcgccga 60 cccgttcgcg
ctggcgcagc acaaatgctc gcgcatcgtg cgtgtggagt accgctgtcc 120
cgagtgcgcc aaggtcttca gctgcccggc caacctggcc tcgcaccgcc gctggcacaa
180 accgcggccc gcgcccgccg ccgcccgcgc gccggagcca gaagcagcag
ccaggctgag 240 gcgcgggagg cacccggcgg cggcagcgac cgggacacgc
cgagccccgg cggcgtgtcc 300 gagtcgggct ccgaggacgg gctctacgag
tgccatcact gcgccaagaa gttccgccgc 360 caggcctacc tacgcaagca
cctgctggcg caccaccagg cgctgcaggc caagggcgcg 420 ccgctagcgc
ccccggccga ggacctactg gccttgtacc ccgggcccga cgagaaggcg 480
ccccaggagg cggccggcga cggcgagggg gccggcgtgc ttgggcctga gtgcgtccgs
540 cgagtgccac cctgtgccca gtgtgcggag agtcgttcgc cagcaaggsc
gctcaggagc 600 rccrcctgcg ccstgctgca cgccgsccag gtgttcccct
gcaagtactg sctcttggca 660 ccttctacag ctcgcccggc cttacgcggc
acatcaacaa gtgccaccca tccgaaaaca 720 gacaggtgat cctcctgcag
gtgcccgtgc gcccggcctg ctagagcgcg ccctccaccc 780 cggcccccga
actgtgcctt cgcttggaga cccacaaaga gagtgcgccc tgcacgcccc 840
gaacccgagt ccgcgctggg ggagcctcgc ccccgccccc accgggtgaa agtgtcgtct
900 ccgcttctct cggtgtggcg tgacggtaac cccatactct ccttttgact
ccttttggaa 960 cccccacttt tacgttgtgt ccctccgcct cccccatggc
gcaacaggag tcagtctctt 1020 tctgtacaag ggagaaaagc tgtacgcgtt
tgtctcgtgg ttggaagcct ccccttggcg 1080 gggagaagct ttttttcttg
ctagtattcg ctgtgttcat ggtctagaaa tgcggtctgg 1140 tctcgcctcg
cctaccaatc tctgctctct atgtatgtag cgtacgggtt gttttgggtg 1200
aatcttgagg aataaatgcc tttatatttc acaggctgta aattgaactt cccacacgat
1260 tagctttatt atggcttgtg aactgctgga gtctggcttt acctttttgt
atgtgaacaa 1320 atcaaattgc ttaaaaaaga gttttcttta gtatagccac
aaatgccttg aactgttgtc 1380 tgggattgtt ttgtgggggg agggaaggga
gtgttccgaa gatgctgtag taactgcctc 1440 agtgtttcac gtaagacttt
ttggtttgat catctttgtt gaggtaggac tatcagttcc 1500 ctctaaatgt
atatgttgat ttatgagtaa ttgttattta ttctttattt atttatatta 1560
attatgaaga ttatgatatt atttgattgc agattttttt ggcgcgctgc cccctcccca
1620 ccctgccact cttgacattc cactgtgcgt tttagaagag agcctttttc
taaagggatc 1680 tgcttaaagt tttaactttt atacctatct gagtgaatta
cagacaacct atcatttatt 1740 ctgcttcgag ggtccccagg gcccttgtac
aaccgacagc tcttactttt aaatgcaatc 1800 tcttttctac atacattatt
ttcttaattg ttagctattt atagaaagct tcaatagaac 1860 tgtttcaact
gtataactat ttactattca aataaaatat tttcaaagtc aaaaaaaaaa 1920
aaaaaaaag 1929 50 6183 DNA Homo sapiens 50 ctttttgtag ggagaagggc
aggatgtttt taactgaatg tgacctcagg ggaatactag 60 agaaaataat
aaaatttctg aatggggcag cgtggagaaa tcctaagaga aatagcataa 120
gagcattttg gaacacatcc aggaaaagat aactttcgac acacctgtag acgttcgcca
180 ggtaaaggag tgatggaaac tctccagttc agatccagta gcttttaggg
aaggaactac 240 agttgctgac ttaagttgaa gaagcatcta tttaatgtct
ggtcaaatcc tacaagaaac 300 acagaaatct atgattaaaa agctgagcac
tttgatatac tgcaaagggt agagaaggca 360 ggacggtaga aattttctgc
aagaaagaat gaatttcagg atttatcact aaataagaca 420 aagtcattta
tttagtcccc ctgacacagc agggcaaact gagttgacat acaagttacc 480
tggagaaaaa gagagcaatt ccaggacttc ctcttcagcc taaaagaagg taccagatct
540 gtgcactggg gcgatgtgga agagacctgc ttattgcccc tgatgtaagc
tccagtaaga 600 aaagacgtca agtacaagta ctaggaaatc actttataca
tctgtttata ggaatgacct 660 caggactttg tgttcatgtt atagatggat
gcagaggctg aagataaaac gctgcgtact 720 cgctctaaag gaaccgaggt
gccaatggat tcactaatcc aggagctcag tgttgcctat 780 gattgctcca
tggcaaagaa gagaacagct gaagatcagg ctttgggggt tccagtcaac 840
aaaaggaaat ccctgctaat gaagccccga cactacagcc caaaagcaga ctgccaagaa
900 gaccgcagtg acaggacaga ggacgatggc cccttggaaa cacatggtca
ctctaccgca 960 gaggaaatca tgataaaacc tatggatgaa agtcttcttt
caactgcaca agaaaactcc 1020 agtaggaagg aagacagata ctcttgttat
caagagctca tggtcaagtc tttaatgcac 1080 ttggggaaat ttgaaaaaaa
tgtatctgtt cagactgtaa gtgaaaattt aaatgacagt 1140 ggcatccagt
ctttaaaagc agagagcgat gaagcagacg agtgctttct gattcattct 1200
gatgatggaa gagacaagat tgatgattct cagccaccct tctgctcctc tgatgacaat
1260 gaaagtaact ctgaaagtgc agaaaatggc tgggacagtg gctccaactt
ctcagaagaa 1320 accaaaccac ctagagtccc aaagtatgtt ttaacagatc
ataaaaaaga cctattggaa 1380 gttcctgaaa taaaaactga aggtgacaaa
tttatccctt gtgagaacag gtgtgattct 1440 gaaacagaaa ggaaagaccc
gcagaatgct ctcgcagaac ccctggatgg caatgcccag 1500 ccctcattcc
ctgacgttga ggaggaagat agcgagagcc tggcagtaat gacggaagag 1560
ggtagtgacc tggaaaaggc caaggggaat ttaagtttgc tggagcaggc aattgctctg
1620 caggctgagc gaggttgtgt tttccataac acctacaaag agctggatag
gttcctgctg 1680 gagcacctag caggggaaag gaggcaaacc aaagttatcg
acatgggtgg aagacaaatc 1740 tttaacaata aacattcacc aaggcctgaa
aagagggaga ccaagtgccc gatccctgga 1800 tgtgatggca cgggacacgt
gacagggctc tacccgcacc accgcagcct ttcggggtgc 1860 ccccacaaag
tgcgggttcc cctggaaatt cttgccatgc atgaaaatgt gctcaagtgt 1920
cccacgccgg gatgcacagg aaggggtcat gtgaacagca accgcaacac ccacaggagt
1980 ctttctggtt gtccaattgc tgcagctgaa aaattggcaa tgtcccagga
taaaaatcag 2040 cttgattctc cccaaactgg gcagtgtcct gaccaggccc
acaggacaag tttggtgaag 2100 caaattgaat tcaatttccc gtcacaagcc
atcacctctc ccagagccac agtgtcaaaa 2160 gaacaagaga agtttggaaa
agtaccattt gattatgcca gttttgatgc ccaagttttc 2220 ggtaaacgcc
ctctcataca aacagtgcaa ggacgaaaaa caccaccatt tcctgaatca 2280
aagcattttc caaatccagt gaaatttcct aatcgactgc ctagtgcagg cgcccacacc
2340 cagagccctg gccgtgccag ctcttatagc tacggtcaat gtagtgaaga
cacccacata 2400 gcagcagctg ctgccatcct gaacctttcc acccgctgca
gggaagccac agacatcctc 2460 tccaacaagc cacagagtct gcatgccaag
ggagccgaaa tagaagtgga tgaaaatggc 2520 acattggact taagcatgaa
aaaaaatcga atcctggaca agtctgcacc cctaacttcc 2580 tctaacactt
ctattccaac tccttcctct tccccattca aaacaagcag cattctggtc 2640
aatgcagcat tctatcaggc tctttgtgac caagagggct gggacactcc tatcaactat
2700 agcaaaactc acgggaagac agaggaggag aaagagaaag acccagtgag
ctctctagaa 2760 aatttagagg aaaaaaagtt tcctggagag gcctctatac
caagccctaa acccaagctt 2820 catgcaagag atctcaaaaa ggaactaatc
acctgtccaa caccaggatg tgatggaagt 2880 ggccacgtga caggaaacta
tgcatctcat cgcagtgttt ctggatgtcc tttagcagat 2940 aagactctaa
aatccctcat ggctgccaac tctcaggagc ttaagtgtcc aaccccaggc 3000
tgcgatggct cggggcacgt gactggaaac tatgcttccc acagaagctt gtccggatgc
3060 cctcgtgcaa ggaaaggtgg tgtcaaaatg acccctacca aggaagaaaa
agaagaccct 3120 gaactgaaat gtcctgtgat agggtgtgat ggccaaggtc
acatatcagg taaatacaca 3180 tcacaccgca cagcttctgg ctgtcctctg
gctgccaaga gacagaagga gaatcctctc 3240 aatggagcct ccctctcctg
gaaactgaac aaacaagagc taccacattg tcccttgcca 3300 ggctgcaatg
ggctgggcca tgtaaataat gtttttgtca cccaccgaag cttatctgga 3360
tgtcctctca atgcacaagt tatcaaaaag ggcaaggttt ctgaagaact catgaccatc
3420 aagctcaaag caactggggg aatagagagt gatgaagaaa ttaggcattt
ggatgaagaa 3480 ataaaggaac tgaatgaatc caaccttaaa attgaagcag
atatgatgaa acttcagacc 3540 cagatcacat ctatggagag caacttaaag
acgatagagg aggagaacaa actcatagaa 3600 cagaacaatg aaagtctgct
gaaagagctg gcaggtctaa gccaagctct catttcaagc 3660 cttgctgaca
tccagcttcc acagatggga cctatcagtg agcagaattt tgaagcatat 3720
gtaaatacac tcacagatat gtacagcaat ctggaacggg actattcccc ggaatgcaaa
3780 gctctactgg aaagtatcaa acaggcagtg aagggtatcc atgtgtagga
tcacagcgct 3840 gccgggcaac agaagttacc aacagcagta aactccagat
ggatctgtta gaggttcatg 3900 tactgctaag gcgtggaggt tgccgtactg
catttacaat ttgcaacatt gcactaattt 3960 tattttcccc agctgatata
aaaaggaaag aaaaactatg atagacttct tggattaaaa 4020 gcaatgcagt
caattattag atcttattta ttttcatatg tttttctttt atttcttcat 4080
tgtactcttc ttttgtaaag tatatgtaaa ataaatgtga catttttata atttatttat
4140 tactaatcaa agagtttttt atcttttaac tgcattttga agtctgccgt
atttttacaa 4200 gtgtgtttat taatttattt tccaatagga tttaaataga
aatgctattc tcaagtcatc 4260 tttcttgctg ggttttaatg aggaaacagg
aaagggtgaa ggaaatcctt gtctaaggac 4320 tgcactatag ttgagtttga
tttttattgc acacttcttc ccccaccttt cactgatttt 4380 tgtatttata
aatgaatttg cggtaaggtg agctgcacgg aaggaataag aagacaaatg 4440
gcgcccacta gtggggaatc cgcactcaca aaagcacagg atgctggaaa acagcctgct
4500 cagaatttgt tagcaataat taaatatagc aatcagcaaa gtattcgact
tggctggacg 4560 gttttcgtta atatgaatta tttatttgaa atgttttaaa
gaaacataag cctttttagt 4620 gatgcagatt tgtctgtttg tttttcaagt
catatcagat cgttggcaac tcgtatccca 4680 agatgaaaaa taagacttgg
tgtgaccagc caggctttcc tgccatatgt tggtacaata 4740 tacaagtgac
aatattggtg tagatttgta cttagcaaat acaaacacat ccaaatgaaa 4800
aattttgtag ataccatatc ccctgaaata gcatttatct tactgggttg actggaaagg
4860 aatggaaaat atagtaacac atgaaaaaat gctactccaa tctgaatgat
tacttcaaac 4920 actggcacct tgggtctcac ccaccatagg aaacaagaca
acattcaatt tgatagaaat 4980 cttgccacaa aacttcaaat gctacaaaat
atacacacac actcacacac acaggcatac 5040 tcacacacag acacacacac
acacacacac acagactcat ccacacttca aattgagccc 5100 acaatcttga
atttctgaac ggatcagagt ttcatagttt ctatagtaaa ggcaatgtct 5160
atttcaggga ttgtaaagta gttaagcatt gtttcaaaag tttttttata tttatttttt
5220 ttaaggaaaa ggtatagaca accagctaaa ctgccttttt ggtgtgcaca
cacatttcat 5280 gtgcagacgt gcctctgtgt aaatgtacac atgaacttca
tgtgggctta attttctgtg 5340 ctataaacaa aagtgtttat tttttattaa
cctcatggat atttagatgg aaagtgatgg 5400 cattcacagg cttgatgtat
tccactgtta ttactgttac ctgcacaaat gaaaaacaat 5460 actcaacagt
aattccactc ccatgaaact ttggtcattg ttatgcatta agtggggctt 5520
atctttggtt tggagttcat ttgaactctt gaaccttagt ttagtgaaga tgaactgtct
5580 gttcttaggt agaaacggtg tttatttaaa aatcagtttt aaaaaatgag
ctaccatatg 5640 tgctgtctat tataaatggg acaccaaaca aaattttcta
ttacagttgt gtacttgcaa 5700 acattttgct atacagtact tcatagatgc
atacaaatga gctcacttat tacaaagaca 5760 aacgtttaat ttgctaaata
ttttaacaag tttgttatat attttattta atttaaaaga 5820 aatctcttac
caacctacat atttattact ataatttgct atgacttcag gttaatttat 5880
ttgtgtttgc atagtttgag caggatgttt tgtgaagtat gtttgtattt atttgcctac
5940 tttgtacttg atgtgttttg taatgtgcac tgaatttgtt ttcttttcaa
ctatgttaat 6000 gatcaatact gtaaattggg tcttttgtaa acaaaaaggc
aatgatgtat gcattttttt 6060 taatttgagg tagtttgttt gtatactgtt
tctccaaaca cttaatattt cttacatcaa 6120 agcaacaaaa ttgtgttcag
tgctgtacat ttggtgtatg gtaggaaata aaaattgata 6180 acg 6183 51 1704
DNA Homo sapiens misc_feature (1)...(1704) n = A,T,C or G 51
tccagaaaaa taaaagatat ataggagcca caagtgtctt ggggaccata taaaacaccg
60 tgtttgggtg cctattagaa tataacgttg ggcctgctgc ctgttacgag
tgtacaatgc 120 cttctcgccg gtttgttcaa tatacccgcc cgcgccgtat
ctttcgcaag gcagtttaca 180 gccctacacc gcaggttacc cagaggtaat
cgggagagct taaaataacc gttactcctg 240 aaaaaaggta tgtaaagagc
gaattttctc agtcatagtt gaataatcaa tgaagtagtc 300 ttgcttccta
atgtccttac ccattcttgg ataattcttt attagaatga atgttgagag 360
cctgggggat cttaggatat tcttgagaaa taaatttgaa gtgccatttt gtgctaaacg
420 taggtagaaa atggcgtttt agattttcaa aagtaaatgg ctaaaaatta
agcattatac 480 ccttcagaaa gtttataagg tttgaccatc atttttttaa
cacagaaatc
tgtttattaa 540 accaaacaaa acagagaaaa ttataccagc cctcaatttt
tgaattttca tttaaataag 600 caaactctaa atccacatct taaaagatgt
ttgtgcagct atgtatttcc aaaatactca 660 tatttcaata agatttttca
cattatattc accaacagta tcacaaaagt tttttttttg 720 ttttttgttt
acataattgt aaggaacagt aattctagaa acactagaag aaaaaagcat 780
agcaatgtcc acagttacaa gaaaaagtgc acattactcg gtcacaatca cagtcattac
840 ttgaaaaact atatgtaaca agtagataag aaatatcact gatgcctcaa
actcattgtc 900 aaaaactgaa tgacataaat tttacatgaa ataaggcaaa
ttcaggaatg cacaaagaat 960 ttgtaatcca accaaakcta aacaacagaa
aaaagttgta taagaagcat gaactaaagt 1020 acttctccct aaatatttaa
aaaataggct tgtctcagtg cacaaagaaa acatcactca 1080 tgtgtatccc
acactataaa ataagaaaga agggtaaagt atgggggata ggagggcaca 1140
gttcattgta agttgcagct gcatccgctg agagttcctt acattatttt tagctagaac
1200 tgaaaattat acaaatcata tcaggagatg taatggtctt tttggaaact
atttctgaaa 1260 gaaatgaaaa gaaaactaca cacaagagtg caaattttca
gattgtcact tgcaacctct 1320 taacattcag tcatctacat ccaggtgctg
ctagagggat gcctggagac agcagcggca 1380 atcaggaacg agcagctcta
agaaaccaag gtgtgatttt ttttcaacaa catgtcttgt 1440 cattattaaa
aaaaaaattc tgggatgaaa actgctatga taaagttgca gtgttgagtg 1500
gggtttttga gatcagcatg agagcagaaa tgcaggcttc tcttggaagt agttcctgat
1560 gtgacgattg aaagaacgta ggcaagggtt tttccagcat caagtgttat
ttttgtagaa 1620 agaatttgga aagaggagaa ggcaaaggga tgtggaaaag
gtacttacag tagtttctca 1680 aaacagtttt cttttaggac ctat 1704 52 1886
DNA Homo sapiens misc_feature (1)...(1886) n = A,T,C or G 52
taaattccgt tgttactcaa gatgactgct tcaagggtaa aagagtgcat cgctttagaa
60 gaagtttggc agtatttaaa tctgttggat cctctcagct atctagtttc
atgggaagtt 120 gctggttttg aatattaagc taaaagtttt ccactattac
agaaattctg aattttggta 180 aatcacactg aaactttctg tataacttgt
attattagac tctctagttt tatcttaaca 240 ctgaaactgt tcttcattag
atgtttattt agaacctggt tctgtgttta atatatagtt 300 taaagtaaca
aataatcgag actgaaagaa tgttaagatt tatctgcaag gatttttaaa 360
aaattgaaac ttgcatttta agtgtttaaa agcaaatact gactttcaaa aaagttttta
420 aaacctgatt tgaaagctaa caattttgat agtctgaaca caagcatttc
acttctccaa 480 gaagtacctg tgaacagtac aatatttcag tattgagctt
tgcatttatg atttatctag 540 aaatttacct caaaagcaga atttttaaaa
ctgcattttt aatcagtgga actcaatgta 600 tagttagctt tattgaagtc
ttatccaaac ccagtaaaac agattctaag caaacagtcc 660 aatcagtgag
tcataatgtt tattcaaagt attttatctt ttatctagaa nccacatatc 720
tatgtccaat ttgatnggga tagtagttag gataactaaa attctgggcc taatttttta
780 aagaatccaa gacaaactaa actttactgg gtatataacc ttctcaatga
ggtaccattc 840 ttttttataa aaaaaattgt tccttgaaat gctaaactta
atggctgtat gtgaaatttg 900 caaaatactg gtattaaaga acgctgcagc
ttttttatgt cactcaaagg ttaatcggag 960 tatctgaaag gaattgtttt
tataaaaaca ttgaagtatt agttacttgc tataaataga 1020 tttttatttt
tgttttttag cctgttatat ttccttctgt aaaataaaat atgtccagaa 1080
gaggcatgtt gtttctagat taggtagtgt cctcatttta tattgtgacc acacagctag
1140 agcaccagag cccttttgct atactcacag tcttgttttc ccagcctctt
ttactagtct 1200 ttcaggaggt ttgctcttag aactggtgat gtaaagaatg
gaagtagctg tatgagcagt 1260 tccaaggcca agccgtggaa tggtagcaat
gggatataat acccttctaa gggaaacatt 1320 tgtatcagta tcatttgatc
tgccatggac atgtgtttaa agtggctttc tggcccttct 1380 ttcaatggct
tcttccctaa aacgtggaga ctctaagtta atgtcgttac tatgggccat 1440
attactaatg cccactgggg tctatgattt ctcaaaattt tcattcggaa tccgaaggat
1500 acagtcttta aactttagaa ttcccaagaa ggctttatta cacctcagaa
attgaaagca 1560 ccatgacttt gtccattaaa aaattatcca tagttttttt
agtgctttta acattccgac 1620 atacatcatt ctgtgattaa atctccagat
ctctgtaaat gatacctaca ttctaaagag 1680 ttaattctaa ttattccgat
atgaccttaa ggaaaagtaa aggaataaat ttttgtcttt 1740 gttgaagtat
ttaatagagt aaggtaaaga agatattaag tccctttcaa aatggaaaat 1800
taattctaaa ctgagaaaaa tgttcctact acctattgct gatactgtct ttgcataaat
1860 gaataaaaat aaactttttt tcttca 1886 53 877 DNA Homo sapiens
misc_feature (1)...(877) n = A,T,C or G 53 ttyggcacga ggaaatttct
aacawtktwt yytttaatag ttagactcat actttatttt 60 gacaaattta
agatagaaaa atatcataat gtgaatatag cagttgctct ttttgtaaca 120
tggtttggga tgtgcagtga aacttgaaag gacttgcttt acaggtggtc cctcttctgg
180 ctgggtttca gttaattctg aattatattc cagccattgc atttgcttga
aagaatattg 240 gacacagtaa aaaaaagaac aggtttggca ttcaataata
aatattataa agcaatgaac 300 caaaacaact tttaaaataa ttactgaaag
caaacttcag acttcatgat taaagctaag 360 aactcatatt ttcaaaatag
ctttaacagt ttctatcaat atataataca atartaggac 420 acttattttt
aaaaaacaag tgagtagaat cagagtaaat atgatatttc agatgactat 480
aaacagtaaa catcaattca atatatttat atatcatttc agcaatatac tctktgccca
540 gctggcgata aaaactgtag ttctatcatc aaaaaatgca tccctgaatg
tcatctttga 600 acttactaag tgctgtcatc atttctacac tccatctttg
gagggggtgg cttagggact 660 cttggtacat gcagatattt agttatggtt
ataatgacaa aaagtaaatg tgccaggagt 720 ctgaagcaga aacgttgcct
tactttgtta agtagcttca cattcttttg tctctgtgat 780 gcctcaggtg
aagtcacact aaataattca cacaggtgct aattttgttg ctctgtgtca 840
gtacctttca gcttctttct tttcttccct tccccac 877 54 1364 DNA Homo
sapiens misc_feature (1)...(1364) n = A,T,C or G 54 tttttttttt
tttttttgat tanattaagg ggctgccagc ccggagaaat acttaagata 60
tgggtgagaa atccccagac ttttatacaa aagatttcca ctttcaaatc aatgtcagta
120 gacattgata aaagtatagc agcatcctct actgaggtga tttcatttat
tccctgcagc 180 ccactgataa atatctcact tctcccaaat agtatgtgga
ctcccagcta agcagaaaac 240 tattgtcatt caactgaaga agaggaagat
aaaagattgt cttgtttcca tcactgtatt 300 acttgtgtaa catgattaca
taattcttat cctaagagaa agctttcata tttaaaaaaa 360 agtcttttca
gataaaatct gcttgtgtct tgaataatat gaaatacaaa ctttcacttt 420
attttattgt aaattatraa gagattattg tcttaaataa tatattgagt tagcttcaag
480 cttcctaaaa tatgaagaga ttgttgtcta aagtcacata ttgacattga
gctcagtggc 540 ctgtttcatc acgtatgtgc tgctacctgt acagcagaca
tgccgctcca gtgacattta 600 taatgacaga agcagggtaa tggtcttgtg
tttgacatga tcagttagga tcatagactt 660 tccctgactc gtagatatta
gccttgaatt gggggaaaag argactttga cacattttag 720 ttattttaat
aacagagatt tactcttttg aaaaataaag gtatctaatg tctccctaat 780
aagtcttctt tccttccaac taaatgacct acacggactt ttattttctt gatcaaagag
840 gtgtttatta aggacttctg gataactata cttttactct atttttaaag
atcacaaagt 900 aattttaaat gtgaacaggt tcccatacca tgaatgctgg
cctcaccttc tctatcatcc 960 acattttgaa atgcaaagaa agctcccttg
taagccatac ttccttcccc actcccatcc 1020 taggatactt gcccagtgct
cattaggcat ttcttattca gatagtccaa atttaggtta 1080 ttatgcttaa
tttgacacat taactaaatg cccagtttta aaatatatcc atcaattcac 1140
gctgaaatgt gcttctttgt gctatcaaat ggaatagaat acacttattt tttaaacaat
1200 cccagaatac tgtgtgtaga cttttgttgt gctcaaataa atgtttactt
atcttacaaa 1260 gctcaaatac tggattgtaa ccatgtgatg aagttatcta
tgttgtacct aacattgcaa 1320 attaatcaat aaatctctgt tgtcaaaaaa
aaaaaaaaaa aaaa 1364 55 539 DNA Homo sapiens misc_feature
(1)...(539) n = A,T,C or G 55 ccgggccccc cctcgagggy ttcaatggtc
agatggaaca gttgaaaggc gcggtcgaaa 60 ccctcgccat cacgatcgcg
caatctggca ttctggaatt cgtcacaacg atcgtcaccg 120 ccttgggcaa
ctttgtcgat aagctcgccg aggtcagccc ggaaactctg aagtgggtca 180
cgatcatcgg tggggtggcg gcggtgctag gtccggtggc gatcggcatc ggcgccgtgg
240 tctctgcgct gggcgccttt ctccctgtca tcgtgcctgt tgcgagcgcc
atcggcgctg 300 tcgtttcggt catcacggcc ggtgccatcc cagccctggc
cgggcttgtt gttgccctat 360 cgcctgtgct cgtgccgctg gcggcggtgg
ctgctgcagt cggcgccgtt tatctggtgt 420 ggaagaactg ggacatgatc
gggcccattc tcgccaagct ttataacgga gtgaagacgt 480 ggctggtcga
taagctcggc aaggtgtggg aaactctcaa gagcaagata aaagccgta 539 56 510
PRT Homo sapiens 56 Met Pro Arg Gly Phe Leu Val Lys Arg Ser Lys Lys
Ser Thr Pro Val 5 10 15 Ser Tyr Arg Val Arg Gly Gly Glu Asp Gly Asp
Arg Ala Leu Leu Leu 20 25 30 Ser Pro Ser Cys Gly Gly Ala Arg Ala
Glu Pro Pro Ala Pro Ser Pro 35 40 45 Val Pro Gly Pro Leu Pro Pro
Pro Pro Pro Ala Glu Arg Ala His Ala 50 55 60 Ala Leu Ala Ala Ala
Leu Ala Cys Ala Pro Gly Pro Gln Pro Pro Pro 65 70 75 80 Gln Gly Pro
Arg Ala Ala His Phe Gly Asn Pro Glu Ala Ala His Pro 85 90 95 Ala
Pro Leu Tyr Ser Pro Thr Arg Pro Val Ser Arg Glu His Glu Lys 100 105
110 His Lys Tyr Phe Glu Arg Ser Phe Asn Leu Gly Ser Pro Val Ser Ala
115 120 125 Glu Ser Phe Pro Thr Pro Ala Ala Leu Leu Gly Gly Gly Gly
Gly Gly 130 135 140 Gly Ala Ser Gly Ala Gly Gly Gly Gly Thr Cys Gly
Gly Asp Pro Leu 145 150 155 160 Leu Phe Ala Pro Ala Glu Leu Lys Met
Gly Thr Ala Phe Ser Ala Gly 165 170 175 Ala Glu Ala Ala Arg Gly Pro
Gly Pro Gly Pro Pro Leu Pro Pro Ala 180 185 190 Ala Ala Leu Arg Pro
Pro Gly Lys Arg Pro Pro Pro Pro Thr Ala Ala 195 200 205 Glu Pro Pro
Ala Lys Ala Val Lys Ala Pro Gly Ala Lys Lys Pro Lys 210 215 220 Ala
Ile Arg Lys Leu His Phe Glu Asp Glu Val Thr Thr Ser Pro Val 225 230
235 240 Leu Gly Leu Lys Ile Lys Glu Gly Pro Val Glu Ala Pro Arg Gly
Arg 245 250 255 Ala Gly Gly Ala Ala Arg Pro Leu Gly Glu Phe Ile Cys
Gln Leu Cys 260 265 270 Lys Glu Glu Tyr Ala Asp Pro Phe Ala Leu Ala
Gln His Lys Cys Ser 275 280 285 Arg Ile Val Arg Val Glu Tyr Arg Cys
Pro Glu Cys Ala Lys Val Phe 290 295 300 Ser Cys Pro Ala Asn Leu Ala
Ser His Arg Arg Trp His Lys Pro Arg 305 310 315 320 Pro Ala Pro Ala
Ala Ala Arg Ala Pro Glu Pro Glu Ala Ala Ala Arg 325 330 335 Ala Glu
Ala Arg Glu Ala Pro Gly Gly Gly Ser Asp Arg Asp Thr Pro 340 345 350
Ser Pro Gly Gly Val Ser Glu Ser Gly Ser Glu Asp Gly Leu Tyr Glu 355
360 365 Cys His His Cys Ala Lys Lys Phe Arg Arg Gln Ala Tyr Leu Arg
Lys 370 375 380 His Leu Leu Ala His His Gln Ala Leu Gln Ala Lys Gly
Ala Pro Leu 385 390 395 400 Ala Pro Pro Ala Glu Asp Leu Leu Ala Leu
Tyr Pro Gly Pro Asp Glu 405 410 415 Lys Ala Pro Gln Glu Ala Ala Gly
Asp Gly Glu Gly Ala Gly Val Leu 420 425 430 Gly Leu Ser Ala Ser Ala
Glu Cys His Leu Cys Pro Val Cys Gly Glu 435 440 445 Ser Phe Ala Ser
Lys Gly Ala Gln Glu Arg His Leu Arg Leu Leu His 450 455 460 Ala Ala
Gln Val Phe Pro Cys Lys Tyr Cys Pro Ala Thr Phe Tyr Ser 465 470 475
480 Ser Pro Gly Leu Thr Arg His Ile Asn Lys Cys His Pro Ser Glu Asn
485 490 495 Arg Gln Val Ile Leu Leu Gln Val Pro Val Arg Pro Ala Cys
500 505 510 57 1047 PRT Homo sapiens 57 Met Asp Ala Glu Ala Glu Asp
Lys Thr Leu Arg Thr Arg Ser Lys Gly 5 10 15 Thr Glu Val Pro Met Asp
Ser Leu Ile Gln Glu Leu Ser Val Ala Tyr 20 25 30 Asp Cys Ser Met
Ala Lys Lys Arg Thr Ala Glu Asp Gln Ala Leu Gly 35 40 45 Val Pro
Val Asn Lys Arg Lys Ser Leu Leu Met Lys Pro Arg His Tyr 50 55 60
Ser Pro Lys Ala Asp Cys Gln Glu Asp Arg Ser Asp Arg Thr Glu Asp 65
70 75 80 Asp Gly Pro Leu Glu Thr His Gly His Ser Thr Ala Glu Glu
Ile Met 85 90 95 Ile Lys Pro Met Asp Glu Ser Leu Leu Ser Thr Ala
Gln Glu Asn Ser 100 105 110 Ser Arg Lys Glu Asp Arg Tyr Ser Cys Tyr
Gln Glu Leu Met Val Lys 115 120 125 Ser Leu Met His Leu Gly Lys Phe
Glu Lys Asn Val Ser Val Gln Thr 130 135 140 Val Ser Glu Asn Leu Asn
Asp Ser Gly Ile Gln Ser Leu Lys Ala Glu 145 150 155 160 Ser Asp Glu
Ala Asp Glu Cys Phe Leu Ile His Ser Asp Asp Gly Arg 165 170 175 Asp
Lys Ile Asp Asp Ser Gln Pro Pro Phe Cys Ser Ser Asp Asp Asn 180 185
190 Glu Ser Asn Ser Glu Ser Ala Glu Asn Gly Trp Asp Ser Gly Ser Asn
195 200 205 Phe Ser Glu Glu Thr Lys Pro Pro Arg Val Pro Lys Tyr Val
Leu Thr 210 215 220 Asp His Lys Lys Asp Leu Leu Glu Val Pro Glu Ile
Lys Thr Glu Gly 225 230 235 240 Asp Lys Phe Ile Pro Cys Glu Asn Arg
Cys Asp Ser Glu Thr Glu Arg 245 250 255 Lys Asp Pro Gln Asn Ala Leu
Ala Glu Pro Leu Asp Gly Asn Ala Gln 260 265 270 Pro Ser Phe Pro Asp
Val Glu Glu Glu Asp Ser Glu Ser Leu Ala Val 275 280 285 Met Thr Glu
Glu Gly Ser Asp Leu Glu Lys Ala Lys Gly Asn Leu Ser 290 295 300 Leu
Leu Glu Gln Ala Ile Ala Leu Gln Ala Glu Arg Gly Cys Val Phe 305 310
315 320 His Asn Thr Tyr Lys Glu Leu Asp Arg Phe Leu Leu Glu His Leu
Ala 325 330 335 Gly Glu Arg Arg Gln Thr Lys Val Ile Asp Met Gly Gly
Arg Gln Ile 340 345 350 Phe Asn Asn Lys His Ser Pro Arg Pro Glu Lys
Arg Glu Thr Lys Cys 355 360 365 Pro Ile Pro Gly Cys Asp Gly Thr Gly
His Val Thr Gly Leu Tyr Pro 370 375 380 His His Arg Ser Leu Ser Gly
Cys Pro His Lys Val Arg Val Pro Leu 385 390 395 400 Glu Ile Leu Ala
Met His Glu Asn Val Leu Lys Cys Pro Thr Pro Gly 405 410 415 Cys Thr
Gly Arg Gly His Val Asn Ser Asn Arg Asn Thr His Arg Ser 420 425 430
Leu Ser Gly Cys Pro Ile Ala Ala Ala Glu Lys Leu Ala Met Ser Gln 435
440 445 Asp Lys Asn Gln Leu Asp Ser Pro Gln Thr Gly Gln Cys Pro Asp
Gln 450 455 460 Ala His Arg Thr Ser Leu Val Lys Gln Ile Glu Phe Asn
Phe Pro Ser 465 470 475 480 Gln Ala Ile Thr Ser Pro Arg Ala Thr Val
Ser Lys Glu Gln Glu Lys 485 490 495 Phe Gly Lys Val Pro Phe Asp Tyr
Ala Ser Phe Asp Ala Gln Val Phe 500 505 510 Gly Lys Arg Pro Leu Ile
Gln Thr Val Gln Gly Arg Lys Thr Pro Pro 515 520 525 Phe Pro Glu Ser
Lys His Phe Pro Asn Pro Val Lys Phe Pro Asn Arg 530 535 540 Leu Pro
Ser Ala Gly Ala His Thr Gln Ser Pro Gly Arg Ala Ser Ser 545 550 555
560 Tyr Ser Tyr Gly Gln Cys Ser Glu Asp Thr His Ile Ala Ala Ala Ala
565 570 575 Ala Ile Leu Asn Leu Ser Thr Arg Cys Arg Glu Ala Thr Asp
Ile Leu 580 585 590 Ser Asn Lys Pro Gln Ser Leu His Ala Lys Gly Ala
Glu Ile Glu Val 595 600 605 Asp Glu Asn Gly Thr Leu Asp Leu Ser Met
Lys Lys Asn Arg Ile Leu 610 615 620 Asp Lys Ser Ala Pro Leu Thr Ser
Ser Asn Thr Ser Ile Pro Thr Pro 625 630 635 640 Ser Ser Ser Pro Phe
Lys Thr Ser Ser Ile Leu Val Asn Ala Ala Phe 645 650 655 Tyr Gln Ala
Leu Cys Asp Gln Glu Gly Trp Asp Thr Pro Ile Asn Tyr 660 665 670 Ser
Lys Thr His Gly Lys Thr Glu Glu Glu Lys Glu Lys Asp Pro Val 675 680
685 Ser Ser Leu Glu Asn Leu Glu Glu Lys Lys Phe Pro Gly Glu Ala Ser
690 695 700 Ile Pro Ser Pro Lys Pro Lys Leu His Ala Arg Asp Leu Lys
Lys Glu 705 710 715 720 Leu Ile Thr Cys Pro Thr Pro Gly Cys Asp Gly
Ser Gly His Val Thr 725 730 735 Gly Asn Tyr Ala Ser His Arg Ser Val
Ser Gly Cys Pro Leu Ala Asp 740 745 750 Lys Thr Leu Lys Ser Leu Met
Ala Ala Asn Ser Gln Glu Leu Lys Cys 755 760 765 Pro Thr Pro Gly Cys
Asp Gly Ser Gly His Val Thr Gly Asn Tyr Ala 770 775 780 Ser His Arg
Ser Leu Ser Gly Cys Pro Arg Ala Arg Lys Gly Gly Val 785 790 795 800
Lys Met Thr Pro Thr Lys Glu Glu Lys Glu Asp Pro Glu Leu Lys Cys 805
810 815 Pro Val Ile Gly Cys Asp Gly Gln Gly His Ile Ser Gly Lys Tyr
Thr 820 825 830 Ser His Arg Thr Ala Ser Gly Cys Pro Leu Ala Ala Lys
Arg Gln Lys 835 840 845 Glu Asn Pro Leu Asn Gly Ala Ser Leu Ser Trp
Lys Leu Asn Lys Gln 850 855 860 Glu Leu Pro His Cys Pro Leu Pro Gly
Cys Asn Gly Leu Gly His Val 865 870 875 880 Asn Asn Val Phe Val Thr
His Arg Ser Leu Ser Gly Cys Pro Leu Asn 885 890 895 Ala Gln Val Ile
Lys Lys Gly Lys Val Ser Glu Glu Leu Met Thr Ile 900 905 910 Lys Leu
Lys Ala Thr Gly Gly Ile Glu Ser Asp Glu Glu Ile Arg His
915 920 925 Leu Asp Glu Glu Ile Lys Glu Leu Asn Glu Ser Asn Leu Lys
Ile Glu 930 935 940 Ala Asp Met Met Lys Leu Gln Thr Gln Ile Thr Ser
Met Glu Ser Asn 945 950 955 960 Leu Lys Thr Ile Glu Glu Glu Asn Lys
Leu Ile Glu Gln Asn Asn Glu 965 970 975 Ser Leu Leu Lys Glu Leu Ala
Gly Leu Ser Gln Ala Leu Ile Ser Ser 980 985 990 Leu Ala Asp Ile Gln
Leu Pro Gln Met Gly Pro Ile Ser Glu Gln Asn 995 1000 1005 Phe Glu
Ala Tyr Val Asn Thr Leu Thr Asp Met Tyr Ser Asn Leu Glu 1010 1015
1020 Arg Asp Tyr Ser Pro Glu Cys Lys Ala Leu Leu Glu Ser Ile Lys
Gln 1025 1030 1035 1040 Ala Val Lys Gly Ile His Val 1045 58 2165
DNA Homo sapiens 58 cgccaccgct gggtgcggcg aggccggcgc gatgcggcag
ctgtgccggg gccgcgtgct 60 gggcatctcg gtggccatcg cgcacggggt
cttctcgggc tccctcaaca tcttgctcaa 120 gttcctcatc agccgctacc
agttctcctt cctgaccctg gtgcagtgcc tgaccagctc 180 caccgcggcg
ctgagcctgg agctgctgcg gcgcctcggg ctcatcgccg tgcccccctt 240
cggtctgagc ctggcgcgct ccttcgcggg ggtcgcggtg ctctccacgc tgcagtccag
300 cctcacgctc tggtccctgc gcggcctcag cctgcccatg tacgtggtct
tcaagcgctg 360 cctgcccctg gtcaccatgc tcatcggcgt cctggtgctc
aagaacggcg cgccctcgcc 420 aggggtgctg gcggcggtgc tcatcaccac
ctgcggcgcc gccctggcag gagccggcga 480 cctgacgggc gaccccatcg
ggtacgtcac gggagtgctg gcggtgctgg tgcacgctgc 540 ctacctggtg
ctcatccaga aggccagcgc agacaccgag cacgggccgc tcaccgcgca 600
gtacgtcatc gccgtctctg ccaccccgct gctggtcatc tgctccttcg ccagcaccga
660 ctccatccac gcctggacct tcccgggctg gaaggacccg gccatggtct
gcatcttcgt 720 ggcctgcatc ctgatcggct gcgccatgaa cttcaccacg
ctgcactgca cctacatcaa 780 ttcggccgtg accacctctc tgttcattgc
cggcgtggtg gtgaacaccc tgggctctat 840 catttactgt gtggccaagt
tcatggagac cagaaagcaa agcaactacg aggacctgga 900 ggcccagcct
cggggagagg aggcgcagct aagtggagac cagctgccgt tcgtgatgga 960
ggagctgccc ggggagggag gaaatggccg gtcagaaggt ggggaggcag caggtggccc
1020 cgctcaggag agcaggcaag aggtcagggg cagcccccga ggagtcccgc
tggtggctgg 1080 gagctctgaa gaagggagca ggaggtcgtt aaaagatgct
tacctcgagg tatggaggtt 1140 ggttagggga accaggtata tgaagaagga
ttatttgata gaaaacgagg agttacccag 1200 tccttgagaa ggaggtgcat
gtacgtacct atgtgcatac acttatttta tatgttagaa 1260 atgacgtgtt
ttaatgagag gcctccccgt tttattcttt gaggagtggg gaagggaaga 1320
aaagaaagaa gctgaaaggt actgacacag agcaacaaaa ttagcacctg tgtgaattat
1380 ttagtgtgac ttcacctgag gcatcacaga gacaaaagaa tgtgaagcta
cttaacaaag 1440 taaggcaacg tttctgcttc agactcctgg cacatttact
ttttgtcatt ataaccataa 1500 ctaaatatct gcatgtacca agagtcccta
agccaccccc tccaaagatg gagtgtagaa 1560 atgatgacag cacttagtaa
gttcaaagat gacattcagg gatgcatttt ttgatgatag 1620 aactacagtt
tttatcgcca gctgggcaaa gagtatattg ctgaaatgat atataaatat 1680
attgaattga tgtttactgt ttatagtcat ctgaaatatc atatttactc tgattctact
1740 cacttgtttt ttaaaaataa gtgtcctact attgtattat atattgatag
aaactgttaa 1800 agctattttg aaaatatgag ttcttagctt taatcatgaa
gtctgaagtt tgctttcagt 1860 aattatttta aaagttgttt tggttcattg
ctttataata tttattattg aatgccaaac 1920 ctgttctttt ttttactgtg
tccaatattc tttcaagcaa atgcaatggc tggaatataa 1980 ttcagaatta
actgaaaccc agccagaaga gggaccacct gtaaagcaag tcctttcaag 2040
tttcactgca catcccaaac catgttacaa aaagagcaac tgctatattc acattatgat
2100 atttttctat cttaaatttg tcaaaataaa gtatgagtct aactattaaa
aaaaaaaaaa 2160 aaaaa 2165 59 1176 DNA Homo sapiens 59 atgcggcagc
tgtgccgggg ccgcgtgctg ggcatctcgg tggccatcgc gcacggggtc 60
ttctcgggct ccctcaacat cttgctcaag ttcctcatca gccgctacca gttctccttc
120 ctgaccctgg tgcagtgcct gaccagctcc accgcggcgc tgagcctgga
gctgctgcgg 180 cgcctcgggc tcatcgccgt gccccccttc ggtctgagcc
tggcgcgctc cttcgcgggg 240 gtcgcggtgc tctccacgct gcagtccagc
ctcacgctct ggtccctgcg cggcctcagc 300 ctgcccatgt acgtggtctt
caagcgctgc ctgcccctgg tcaccatgct catcggcgtc 360 ctggtgctca
agaacggcgc gccctcgcca ggggtgctgg cggcggtgct catcaccacc 420
tgcggcgccg ccctggcagg agccggcgac ctgacgggcg accccatcgg gtacgtcacg
480 ggagtgctgg cggtgctggt gcacgctgcc tacctggtgc tcatccagaa
ggccagcgca 540 gacaccgagc acgggccgct caccgcgcag tacgtcatcg
ccgtctctgc caccccgctg 600 ctggtcatct gctccttcgc cagcaccgac
tccatccacg cctggacctt cccgggctgg 660 aaggacccgg ccatggtctg
catcttcgtg gcctgcatcc tgatcggctg cgccatgaac 720 ttcaccacgc
tgcactgcac ctacatcaat tcggccgtga ccacctctct gttcattgcc 780
ggcgtggtgg tgaacaccct gggctctatc atttactgtg tggccaagtt catggagacc
840 agaaagcaaa gcaactacga ggacctggag gcccagcctc ggggagagga
ggcgcagcta 900 agtggagacc agctgccgtt cgtgatggag gagctgcccg
gggagggagg aaatggccgg 960 tcagaaggtg gggaggcagc aggtggcccc
gctcaggaga gcaggcaaga ggtcaggggc 1020 agcccccgag gagtcccgct
ggtggctggg agctctgaag aagggagcag gaggtcgtta 1080 aaagatgctt
acctcgaggt atggaggttg gttaggggaa ccaggtatat gaagaaggat 1140
tatttgatag aaaacgagga gttacccagt ccttga 1176 60 1089 DNA Homo
sapiens 60 cgccaccgct gggtgcggcg aggccggcgc gatgcggcag ctgtgccggg
gccgcgtgct 60 gggcatctcg gtggccatcg cgcacggggt cttctcgggc
tccctcaaca tcttgctcaa 120 gttcctcatc agccgctacc agttctcctt
cctgaccctg gtgcagtgcc tgaccagctc 180 caccgcggcg ctgagcctgg
agctgctgcg gcgcctcggg ctcatcgccg tgcccccctt 240 cggtctgagc
ctggcgcgct ccttcgcggg ggtcgcggtg ctctccacgc tgcagtccag 300
cctcacgctc tggtccctgc gcggcctcag cctgcccatg tacgtggtct tcaagcgctg
360 cctgcccctg gtcaccatgc tcatcggcgt cctggtgctc aagaacggcg
cgccctcgcc 420 aggggtgctg gcggcggtgc tcatcaccac ctgcggcgcc
gccctggcag gagccggcga 480 cctgacgggc gaccccatcg ggtacgtcac
gggagtgctg gcggtgctgg tgcacgctgc 540 ctacctggtg ctcatccaga
aggccagcgc agacaccgag cacgggccgc tcaccgcgca 600 gtacgtcatc
gccgtctctg ccaccccgct gctggtcatc tgctccttcg ccagcaccga 660
ctccatccac gcctggacct tcccgggctg gaaggacccg gccatggtct gcatcttcgt
720 ggcctgcatc ctgatcggct gcgccatgaa cttcaccacg ctgcactgca
cctacatcaa 780 ttcggccgtg accacctctc tgttcattgc cggcgtggtg
gtgaacaccc tgggctctat 840 catttactgt gtggccaagt tcatggagac
cagaaagcaa agcaactacg aggacctgga 900 ggcccagcct cggggagagg
aggcgcagct aagtggagac cagctgccgt tcgtgatgga 960 ggagctgccc
ggggagggag gaaatggccg gtcagaaggt ggggaggcag caggtggccc 1020
cgctcaggag agcaggcaag aggtcagggg cagcccccga ggagtcccgc tggtggctgg
1080 gagctctga 1089 61 362 PRT Homo sapiens 61 Arg His Arg Trp Val
Arg Arg Gly Arg Arg Asp Ala Ala Ala Val Pro 5 10 15 Gly Pro Arg Ala
Gly His Leu Gly Gly His Arg Ala Arg Gly Leu Leu 20 25 30 Gly Leu
Pro Gln His Leu Ala Gln Val Pro His Gln Pro Leu Pro Val 35 40 45
Leu Leu Pro Asp Pro Gly Ala Val Pro Asp Gln Leu His Arg Gly Ala 50
55 60 Glu Pro Gly Ala Ala Ala Ala Pro Arg Ala His Arg Arg Ala Pro
Leu 65 70 75 80 Arg Ser Glu Pro Gly Ala Leu Leu Arg Gly Gly Arg Gly
Ala Leu His 85 90 95 Ala Ala Val Gln Pro His Ala Leu Val Pro Ala
Arg Pro Gln Pro Ala 100 105 110 His Val Arg Gly Leu Gln Ala Leu Pro
Ala Pro Gly His His Ala His 115 120 125 Arg Arg Pro Gly Ala Gln Glu
Arg Arg Ala Leu Ala Arg Gly Ala Gly 130 135 140 Gly Gly Ala His His
His Leu Arg Arg Arg Pro Gly Arg Ser Arg Arg 145 150 155 160 Pro Asp
Gly Arg Pro His Arg Val Arg His Gly Ser Ala Gly Gly Ala 165 170 175
Gly Ala Arg Cys Leu Pro Gly Ala His Pro Glu Gly Gln Arg Arg His 180
185 190 Arg Ala Arg Ala Ala His Arg Ala Val Arg His Arg Arg Leu Cys
His 195 200 205 Pro Ala Ala Gly His Leu Leu Leu Arg Gln His Arg Leu
His Pro Arg 210 215 220 Leu Asp Leu Pro Gly Leu Glu Gly Pro Gly His
Gly Leu His Leu Arg 225 230 235 240 Gly Leu His Pro Asp Arg Leu Arg
His Glu Leu His His Ala Ala Leu 245 250 255 His Leu His Gln Phe Gly
Arg Asp His Leu Ser Val His Cys Arg Arg 260 265 270 Gly Gly Glu His
Pro Gly Leu Tyr His Leu Leu Cys Gly Gln Val His 275 280 285 Gly Asp
Gln Lys Ala Lys Gln Leu Arg Gly Pro Gly Gly Pro Ala Ser 290 295 300
Gly Arg Gly Gly Ala Ala Lys Trp Arg Pro Ala Ala Val Arg Asp Gly 305
310 315 320 Gly Ala Ala Arg Gly Gly Arg Lys Trp Pro Val Arg Arg Trp
Gly Gly 325 330 335 Ser Arg Trp Pro Arg Ser Gly Glu Gln Ala Arg Gly
Gln Gly Gln Pro 340 345 350 Pro Arg Ser Pro Ala Gly Gly Trp Glu Leu
355 360 62 391 PRT Homo sapiens 62 Met Arg Gln Leu Cys Arg Gly Arg
Val Leu Gly Ile Ser Val Ala Ile 5 10 15 Ala His Gly Val Phe Ser Gly
Ser Leu Asn Ile Leu Leu Lys Phe Leu 20 25 30 Ile Ser Arg Tyr Gln
Phe Ser Phe Leu Thr Leu Val Gln Cys Leu Thr 35 40 45 Ser Ser Thr
Ala Ala Leu Ser Leu Glu Leu Leu Arg Arg Leu Gly Leu 50 55 60 Ile
Ala Val Pro Pro Phe Gly Leu Ser Leu Ala Arg Ser Phe Ala Gly 65 70
75 80 Val Ala Val Leu Ser Thr Leu Gln Ser Ser Leu Thr Leu Trp Ser
Leu 85 90 95 Arg Gly Leu Ser Leu Pro Met Tyr Val Val Phe Lys Arg
Cys Leu Pro 100 105 110 Leu Val Thr Met Leu Ile Gly Val Leu Val Leu
Lys Asn Gly Ala Pro 115 120 125 Ser Pro Gly Val Leu Ala Ala Val Leu
Ile Thr Thr Cys Gly Ala Ala 130 135 140 Leu Ala Gly Ala Gly Asp Leu
Thr Gly Asp Pro Ile Gly Tyr Val Thr 145 150 155 160 Gly Val Leu Ala
Val Leu Val His Ala Ala Tyr Leu Val Leu Ile Gln 165 170 175 Lys Ala
Ser Ala Asp Thr Glu His Gly Pro Leu Thr Ala Gln Tyr Val 180 185 190
Ile Ala Val Ser Ala Thr Pro Leu Leu Val Ile Cys Ser Phe Ala Ser 195
200 205 Thr Asp Ser Ile His Ala Trp Thr Phe Pro Gly Trp Lys Asp Pro
Ala 210 215 220 Met Val Cys Ile Phe Val Ala Cys Ile Leu Ile Gly Cys
Ala Met Asn 225 230 235 240 Phe Thr Thr Leu His Cys Thr Tyr Ile Asn
Ser Ala Val Thr Thr Ser 245 250 255 Leu Phe Ile Ala Gly Val Val Val
Asn Thr Leu Gly Ser Ile Ile Tyr 260 265 270 Cys Val Ala Lys Phe Met
Glu Thr Arg Lys Gln Ser Asn Tyr Glu Asp 275 280 285 Leu Glu Ala Gln
Pro Arg Gly Glu Glu Ala Gln Leu Ser Gly Asp Gln 290 295 300 Leu Pro
Phe Val Met Glu Glu Leu Pro Gly Glu Gly Gly Asn Gly Arg 305 310 315
320 Ser Glu Gly Gly Glu Ala Ala Gly Gly Pro Ala Gln Glu Ser Arg Gln
325 330 335 Glu Val Arg Gly Ser Pro Arg Gly Val Pro Leu Val Ala Gly
Ser Ser 340 345 350 Glu Glu Gly Ser Arg Arg Ser Leu Lys Asp Ala Tyr
Leu Glu Val Trp 355 360 365 Arg Leu Val Arg Gly Thr Arg Tyr Met Lys
Lys Asp Tyr Leu Ile Glu 370 375 380 Asn Glu Glu Leu Pro Ser Pro 385
390 63 442 DNA Homo sapiens misc_feature 220,391,428 n = A,T,C or G
63 atagtaagca ctgatgtgtt tattcgatga aataggggtg ggggtgtagc
agccctagtc 60 ccacattgca tgggctggtg actgagttaa cagcaaagtg
ggatgcaaaa ggttcctgat 120 tggagacccc cggattcggg ttctggattt
gctggccact tactctatga cttggggcat 180 gtcactgtca tggcctcagt
ttccccttct gcacagtgtn ttattggata gttccagctc 240 tgacatgcta
ggattatgtg atactgtcaa tcaagactag ggttggccta agcacatggt 300
ctgaaaacac ctcgggctca tggacatatt ttctccgcat ggggagtggg cagctgctga
360 gtggcaaggc tgccctccaa agctgtccat nccacgcccg gggtgctgtg
ggtctccttt 420 ccctcgtngc cgaattcttg gg 442 64 456 DNA Homo sapiens
64 cttcaaccat aaaaacaaag ggctctgatt gctttagggg ataagtgatt
taatatccac 60 aaacgtcccc actcccaaaa gtaactatat tctggatttc
aacttttctt ctaattgtga 120 atccttctgt tttttcttct taaggaggaa
agttaaagga cactacaggt catcaaaaac 180 aagttggcca aggactcatt
acttgtctta tatttttact gccactaaac tgcctgtatt 240 tctgtatgtc
cttctatcca aacagacgtt cactgccact tgtaaagtga aggatgtaaa 300
cgaggatata taactgtttc agtgaacaga ttttgtgaag tgccttctgt tttagcactt
360 taagtttatc acattttgtt gacttctgac attccacttt cctaggttat
aggaaagatc 420 tgtttatgta gtttgttttt aaaatgtgcc aatgcc 456 65 654
DNA Homo sapiens 65 aataaattcc agccttctct ttcttgctgc ttcctcagat
attttcctcc tttcttctcc 60 agtattcact ctcttctctg gagtttgatg
ggcctgttta tgtttttgca gtggtttctt 120 ttcgtgtaat tttttatctc
catatttctt atatgctaaa ggtattccat atttagcggc 180 aggctttgta
attttctgag caggcataac agaaatcgag ttttgtcctg aagctggtct 240
tttagctggt ataggctgtg atccaaactt cgaaaatgtt tttagacaaa attcttctgc
300 aataagctga ggagagagaa acttttcaat gcgtttggct ataaaacctt
tctccaatat 360 ggagttgact gatggtctat ccctaggatt tcttttaaat
aactgagaca ccaaactgcg 420 gagatcatag gaataatgca aagacacagg
tggaaaagat ccagatatta tcttcagtac 480 caggtttttc atactgccag
cttcaaaagc atgtttaagt gtacacagct cataaaggac 540 acaccccaga
gcccaaatgt ccttttatta ttgtaagttt gttttcacag atttcaggtg 600
acaagtagat tggggcccct atcaagttcg gccccctctc cagtctttta gaac 654 66
592 DNA Homo sapiens 66 tttttttttt tttttttatt gggaataaat ttatcaaaaa
acatgtcatc caattcccac 60 aaatgagaca ttttaaatac agaatacact
ctgttcatga atataaaatc cccaggtgaa 120 agtcccttaa aacactatta
tggttatgtt tcctagaata attttataac tttttcagag 180 aattccttta
aacttgttaa aataccttgt tgctagtgct cagaacatct aggttcagtc 240
tttattttta agacagtatc tatcctaggc aaatgagagc ttgtttttat gtatttaaga
300 gtttcctctt gtcatttcaa tgtcaaattg atttgactca atttcatgat
ttcatctcgc 360 tcaaggccat caaccggtca gagccagagc ccttcaaagg
ctgtatgtga gtatatgagg 420 gaaaactttc cacataattt tacatcattt
ctatctcata gcagttttag ttttctcata 480 gctatctcat agcagtttta
gttttctcaa attctatgct gtttttgtac tactgcagct 540 gaccaatcca
aagccagttt acactcagca tgtgttattc tactttaaaa ta 592 67 469 DNA Homo
sapiens misc_feature 245,298,314,339,424,440,465 n = A,T,C or G 67
gatgccaaaa atgctttccc aagtggctaa cattctgtat tcccaccagc aatatatgag
60 agattaagtt gcttttcaaa cccatttatg ctcagtattg tcaggttttg
ttttgttctg 120 ggttctttat ttgttggttt tcttttttat ttcagccatg
ctaataggtg tgattgtggt 180 tttaatttgc aattccctaa cttcataaat
tagggaacac agaacacaca tatgacacag 240 aaaantgcat ttgacctgat
tttacttcct actattaaga aacagataaa attcatantg 300 tccctggaac
accntttttt tgttgcttta tttgtcatna catttaatct tttgttaagt 360
ggaaatggtc tcttcagata atttttttcc attttaaatc aggttggttg acctatacat
420 tgtngttttg agagttccan aaggtatccc gtattccaaa tcctncatt 469 68
510 DNA Homo sapiens misc_feature 424,462 n = A,T,C or G 68
tttttcctga gaatttaatt ttatttgctg tagattcaaa atgaggaagt ggtaaatgca
60 ttatttactc aaagcataaa gtcagcctta ggtaggagat gtaacaactc
ctcaacttta 120 cactatccag ttaaagccaa tttttaaaac cttttttttc
cttatgatga cccttgagtc 180 atagaaaact tttcatttta gaaaatgtta
agcatgaaca caaaaagact acgataacag 240 tgttataaac actcgtgtac
ccaaggccca gctttaacat tcatcactta gcatgtttaa 300 ggtagtgctt
aggttgaaat ttatattgtg tgtatcagaa taaagagcag ttcttgcaga 360
tagctagaat tacttcattt ttataggagt ttagagcata aactaacaag ggaatctagg
420 cccnttatag taaatatcct aaaagcattt taattttaca gnattggaca
gcggtatgcc 480 atggacctat tcccatttgg tcaggggcaa 510 69 483 DNA Homo
sapiens 69 tgcatcagtt aatgtaatca gcccacagga tggggattga atggaagtat
gcccagtacc 60 tttaagatat gaagctggtc tgaagtacac cttgaacaat
atatgtacag ttcatcacac 120 actgtattta tttgctggag tgtaaattct
cggagaacag aatttaagac ttggggcaaa 180 cagagtctct tttctcctcc
aacttgaaaa caagaaatag attccccttc caacacagtc 240 tgagtgagtt
ctgtggagct atctgaaggg atgagcaatg ggccaggaag aacctgaggt 300
gatggaagag gcagaaatac agtaggcgac atgctttctt gggaatgccg agcagaaaat
360 gctgctggtc caccagcgag ctctgactac tttaatggaa ttgtgccatg
tgtgtttcaa 420 actgggatta aatggcaatt ttagggaacg agtacaggtc
gcctacatgg ctccatcagt 480 ttc 483 70 481 DNA Homo sapiens 70
gtactggaca gacgtgagcg aggaggccat caagcagacc tacctgaacc agacgggggc
60 cgccgtgcag aacgtggtca tctccggcct ggtctctccc gacggcctcg
cctgcgactg 120 ggtgggcaag aagctgtact ggacggactc agagaccaac
cgcatcgagg tggccaacct 180 caatggcaca tcccggaagg tgctcttctg
gcaggacctt gaccagccga gggccatcgc 240 cttggacccc gctcacgggt
acatgtactg gacagactgg ggtgagacgc cccggattga 300 gcgggcaggg
atggatggca gcacccggaa gatcattgtg gactcggaca tttactggcc 360
caatggactg accatcgacc tggaggagca gaagctctac tgggctgacg ccaagctcag
420 cttcatccac cgtgccaacc tggacggctc gttccggcag aaggtggtgg
agggcagcct 480 g 481 71 341 DNA Homo sapiens 71 cggccgcggc
gaggctggag aagtagtgct ggccgggcga gtcgctccag caggccgggg 60
acgcgggcgc ggcagggggc gtggggcccg gctctggtgg ggggtcctgg gcccgcacat
120 agctgcgaag ggtgatgtcg gccgagcccc ctgactccag tgggatgggg
tgtgtgtgga 180 agtggcggag catgtcaagc acagactgga accacagatg
ctgtacgtga cactggccgt 240 ggccgttcag ggacaggcgc atgtgcttgg
ccttgccctg gaagttgaag gtcagcacgt
300 actccccagg ccgagtctca ctttggcggc ccctcgtgcc g 341 72 283 DNA
Homo sapiens 72 tttttagatc catccattta ttccttcagc caacattttc
tgggattcct tgtgtgctag 60 gcctcgtgcc accatctgga gatgcagaga
ggcgggagac ccatgtggcc tttgaggggc 120 tttcaggctc gtgggggttc
aggcacagac accaccaatc tgaaccaggg gactgcagga 180 tgctgggtta
ggggagagag ggataggctg gctggcctag ggggtcctca ggaagtcttt 240
gggggtaagg agagaactcc tgaaaggtaa ggagaagccg agg 283 73 485 DNA Homo
sapiens 73 ttttttttat ttttaggata ttttatttta atgcaaatga aatttctatc
tatgtgaaac 60 tggtaaaggg gagatatagg aactcctatt tttctctctg
tcttcctctc tgtttcttct 120 ttttttattt atttttggat tatagatgct
cctctcagtt gcaagttgca atgctccaca 180 tctctcagcc agcacctggc
tctgttccag ggcttttagt gagtgctctc tgtcaaggca 240 tgaataatac
agcccctagg ctgttggcag actccaaatg aggcgtgcat acatcaggaa 300
gcaagccctt gactttagct ccagaacagc ctccttctgt gtcttgcata tttgccactg
360 acatgaccac tgccgtcaca gccaggggtg ggacagctga acagctcttg
tatggctggt 420 tccacgggaa ctcgaacccc tttggaccgc gtgcgatgcc
gcttctcctc ggtgtgcaac 480 tccat 485 74 338 DNA Homo sapiens 74
ttttttgatt atttcagaga tttattgcaa gttaattgtc tgtgaagctg gatattcctt
60 aacatgaagg taataaactt taacgttcca ctcaaaaaga caaaaaccaa
acaacgaaaa 120 ataagaaatt aaccagaaag ctatagcttg ttttcttact
cagaaaaaaa gtataactga 180 taaggtacaa tttctgtaac tggatatttt
tcaaaattat aaggctttta gttctaaaag 240 tataaagaac tgtgatgcac
ttctagtcaa cctaatcttg ctagaagctt tatcaacact 300 gacagtctca
atactttctc ttttgctatt atatagtc 338 75 334 DNA Homo sapiens
misc_feature 265 n = A,T,C or G 75 agcggccgcg gcggagcagc aacagttcta
cctgctcctg ggaaacctgc tcagccccga 60 caatgtggtc cggaaacagg
cagaggaaac ctatgagaat atcccaggcc agtcaaagat 120 cacattcctc
ttacaagcca tcagaaatac aacagctgct gaagaggcta gacaaatggc 180
cgccgttctc ctaagacgtc tcttgtcctc tgcatttgat ggaagtctat ccagcacttc
240 cctcttgatg ttcagactgc catcnagagt gagctactca tgaattattc
agatggaaac 300 acaatctagc atgaggaaaa aaggtttgtg atat 334 76 248 DNA
Homo sapiens misc_feature 32,33 n = A,T,C or G 76 gataggcata
aacgtgttta ttaagtgaaa cnnatccttt aaaaataaaa aagggaagcc 60
tgtatataaa tgaagttgtg gattcaacta gccagaattt attctgactt gcaccaaacc
120 acacaaaatc ttttaaaagt ctagttagtc gtagtctaaa tggacactcc
agagtctgtt 180 cttgaattcc attgcaagag ctccaacttc ctactttcag
aagggatggg gatcaagatg 240 agggttgt 248 77 515 DNA Homo sapiens
misc_feature 395,476 n = A,T,C or G 77 atgtagaaac agcatcaagc
tgtttctctc taccgtcttt gatagaaata aaaataaaaa 60 taaaaagttg
aattgcagaa aagctaagag gtttttagtt tttgtttttt gttttccttc 120
caccagtcaa ttattggaaa ggatttagtg agtctggttt attttagctt caatctgggt
180 ttgtacacaa gcaaaaagca aatgttgaat tttcaggtag accttcatgc
agacatgcaa 240 aaccaactgt ctcggtggtg aggagccatg gggagctctc
cgaagggctt tccaggcagt 300 gggctaatgg gcaaaatgac tactcagtgg
ccctgctgac cgatggtaac ggtgtgccaa 360 ggatatctat cagcccatct
gagaatatga aacanagtgc tgagattcta cttacctaag 420 taacaaagaa
accgtaagca acacgactga cagccagaag ggaacactgg aatggngggg 480
tgaatggtgt cctgattagc accccccaat ctcgc 515 78 532 DNA Homo sapiens
78 cctgttgtta tatagtttat tactgtcata gctaagaaaa ggcagtcgat
ttcaacataa 60 tccatatcta tgttcaaatt ctcaaactat aggatatcta
tgtttcaaat tgtaatttat 120 aacctggtaa gtattctaaa caaaatattg
acaatccatt agctgaccta aaatcttatg 180 aagctgtatc atcagtttaa
caaatacaca cgactttagc aaaagtatat acagatagta 240 tttataatac
ttataataca ggcatggact aaaaaataca gataaaattg gagcaaatta 300
aaagaggagt tgcattcaaa atattttttc catttgatat cattagaatt acaaaagcag
360 taataaaaaa atctaatgtt aaggcaatga caaataacaa agataacagt
tgcccaagga 420 gcgaggggtt gggaggtgaa tgcacaatca aggaggggca
caaaacagcc ttcaggttaa 480 tttgttttat taagggggga gtcattggta
gatagtcttt acatcttttt at 532 79 431 DNA Homo sapiens 79 gggataagca
aaatgagtcc aacctttatt ctgataatag ccagtaaatt tgcaaagaga 60
ggagacaaac tgtaattgta tacataaaaa cacctagtcc cactttaaaa ttttaatatc
120 tatatatagt actgtattta atttttaaag atgaagacag caaaaatatt
cacattaaaa 180 tatcttacag aaatcattat tcttctattc aagaaaacca
attatactaa gttaacaggg 240 aaaatttaac agaggaaatt ctccttggga
cacttattga actgaggatt tcacttcata 300 gtttaaaaaa gtaaacaggt
ctcaggtgtc tttttcatgg gtaggtcacc ttatcaatct 360 gaattacagt
tcatgggtaa agctaacttt ttttgtgtga aataagttaa taatgccaat 420
tcagtttctt g 431 80 431 DNA Homo sapiens misc_feature 361,431 n =
A,T,C or G 80 acaaaccttc cgggggttgc ctgagtggct gctctcggaa
aagcggatcc taaataaagc 60 gggagggtta tagggcgacg tcgaggagag
gacaggtctc gagtcactgc tacagtttca 120 ggtcactggg ctccgcagca
gatcgtgttt tctcccgtgg ctcgagagct gcgctggttt 180 ctcatgcaaa
ctcagagccg agctaatgac atgagcaact tttactttta cacaagatga 240
gcacgcgtgc cgaggcgctg ggcggcggct gtgtgagttg gtggcccaga cgaacagctt
300 gtgcgagact ctgggcattt cggtttctag atacaagatt tgcttaaatg
tcacagtcca 360 nagaagtgga tttcagtcat tgtagctact ggatgcacac
aaagtaaaaa aaaaaaaact 420 tcacttgccg n 431 81 471 DNA Homo sapiens
81 aaggtcagat attgtttaac acttgaaatt ccaaagagaa aaaatattcc
caatgagtgc 60 tctgtttcct atagagtaat tgctgaaata aaggaacaca
gaaaacaagg cttctgccag 120 ttgtcactta caaaaacata cagaggatca
taatctagag acatggctaa ggcctcaggt 180 ggtttcatgc tcaagattga
tgttttgcca gagagctgag ttgtggagtc ctgtttcgga 240 agggctgtga
tggtggtgac ttcatcctca gctccttgct ttagggctcg ggcaagcttt 300
tgaggtctgt aacttgttga agacttgtgg acagagaatg gctgatatct cttaattttg
360 tacagttgag gaacctgcag attgaagaag gaataactct gcttgatttg
aacttctgaa 420 gacttaattg ggaccagtcc aaggccatca ggagccaact
cgttggagtc c 471 82 450 DNA Homo sapiens 82 tgtcaatttt tgcaaatcaa
agtgtatcat ttctccaatt ctactgatgc cagtttccaa 60 gtccaattac
tttttctacc ttctaatttt tcttaatttc taagccaata tgttaaaaac 120
tattcttttg gctttcacaa tgttgcatta tcctaactgc ctctgatatc ttcaacaatt
180 catttggtct ttaatgaaac tctttccatg taatgctctt tattaaatgt
agatgtttcc 240 ttaagaatga atctgcacca gccctttgct cttctccatg
atttcaccta ctctcacaat 300 ggtgatgggc attcccatgg ccctgacagc
ttactgtatc tctttagcct gatctctccc 360 tagaaatata atgttcatct
gtgtttgtct gatgaggact gcctgatagc tgccaaatca 420 acaaggataa
aaccagaatt cacattccct 450 83 540 DNA Homo sapiens 83 ttatacaaaa
gcatttaaca agcttaaaaa atgaaactca atgaaaaaaa aaagaaggtt 60
tgaacacagt caaataacct gagaagtgac agatggaaaa gcaacagaat gcaagcacct
120 tgtaaggtct gtaatctttg gatttactgt gaaaagtttc agaacatcat
agactcttac 180 tgccacattg tccatagacc ctggaaaata acagtgaaat
tcatatgtat acacatatat 240 atgaatacac actcatgcat gcacactgtc
ttcacacacc cctcctcacc acttaaccgg 300 agttacataa atgcttctca
gatatgtcat tgcatttgtt tgttttctgc atctcaacta 360 agttcagcgg
cttgcgcctg tgacattaat tatgcaagat tcaaacaacc aagcaggcac 420
attttggggg tgagttttaa gaaatctgtg acctgaaaga aattctgtgg ggactgtctg
480 ggttatccag tttattccgt gattatattc tgtttttagg tcttgaccta
tttttaagct 540 84 559 DNA Homo sapiens misc_feature
493,499,506,517,537,550,559 n = A,T,C or G 84 gttgttgctg ctgtttttac
tcggacaatg cttattttac agcggaattg acaaataaag 60 ccttatttta
cacatccgaa gaaacaccat cacaggaggt ttgtaggtcg gctgtgtgct 120
ttccaaaaca gcaaaataga ttcttcccat ccaaccccct ttcctcttgt agagtagggt
180 gtggctcgtg gggcttcgtc tctctgcagg cacagaaact ggcagacctg
gtccctcctg 240 agcgggccct gctcaaggga atggtgccag attttgaaca
caggtaaaca ggctccttca 300 taacaacact gtgcatttct gtgtcatttt
gtttattgct cactgagttg ttgccacctc 360 agctcttggt ggaaaacagt
gggtgtccag aaattgctga cacaagaaga tggattgcct 420 atggtccgtt
agggacacag ggcagcccca gccagatccc actggtccat gcagggcatc 480
gcagtagaaa ctnaacgtnc cacttngtaa caggctncaa gacaccaatt ccggcancat
540 gggaaagaan taaaccttn 559 85 2466 DNA Homo sapiens 85 agttggtccg
agctgccgaa aggtctggtc gcagagacag gaacgtgtaa tcctcagcgt 60
gctccagccc acagcttcgc tctactgctc ggcagggcag ctggcctctg ggcaccggcg
120 gcccctctgc ctcgcggaaa agcctgatga agtcctccga tattgatcag
gatttattca 180 cagacagtta ctgcaaggtg tgcagtgcac agctgatctc
cgaatcgcag cgtgtggccc 240 actacgagag tcgaaaacat gcaagcaaag
tccgactgta ttacatgctt caccccaggg 300 atggagggtg tcctgccaag
aggctccggt cagaaaatgg aagtgatgcc gacatggtgg 360 ataagaacaa
gtgctgcaca ctctgcaaca tgtcattcac ttcagcggtg gtggccgatt 420
cccattatca aggcaaaatc cacgccaaga ggttaaaact cttgctagga gagaagaccc
480 cattaaagac cacagcaaca cccctgagcc cacttaagcc cccacggatg
gacactgctc 540 cggtggtcgc atctccctat caaagaagag attcagacag
atactgtggg ctctgtgcag 600 cctggtttaa taaccctctg atggcccagc
aacattatga tggcaagaaa cacaaaaaga 660 atgcggcaag agttgctttg
ttagaacaac tggggacaac cctggatatg ggggaactga 720 gaggtctgag
gcgcaattac agatgtacca tctgcagtgt ctccctaaac tcaatagaac 780
agtatcatgc ccatctgaaa ggatctaaac accagaccaa cctgaagaat aagtagtgaa
840 agcatcaatc aagacataag aacaaaacat tagcatttct ctgccgtgga
gaattgctta 900 tcaaccacca gaggaggctt ctttcttgaa caataaacat
ttcttataag gattcacaga 960 ttcacatacg actgatcttg atttttggaa
atgaatgagg tttctttttt ctttttcctt 1020 tttttaattt tggggtaagt
tatgatattt ggatggattt ttaaattctt tcctgataac 1080 atatttagca
catgttctaa attataatcc tatagcaaac agttggagca ttattcaaac 1140
tgaaagtgga aaaatttaaa tttccaattt attctagatt tcctcagagc ataattattc
1200 tgttaaatcc tcaatgagtg tgatgtaaac cacctctatc cagaaatata
cattcttttc 1260 tcatcatgtt ggacacagtt gagggtgaca tgcacagaac
tggaacagat cactattagt 1320 ggaaaatacc aaatggacaa ataaatacca
gtcgttttct ccgttctcca agcacaggag 1380 ccaggtttac catctgaaca
atgaagacga agggagtaaa taaaggaaga attctcatct 1440 tttttcctga
tcattcaaag aacagtttct caaggttaag ccaagtcctc cttgcaagtt 1500
gccaaataat agcttaggaa aagaattagt ctgcctgcat gatgatcttc ttaggcaaaa
1560 acgtcttcac agcccttgac cttggtgaat ttttttcccc aaaagcatcc
aaaagaagaa 1620 ttataaaccc cagaacgaga tggaaataaa caagtatttt
ttttttatga tgtttggcct 1680 gaactgtggg ctttaattgg gggatactga
tcgtttggaa agaagtgaga aaattctgaa 1740 gaaatggcgg ccttgggcta
ggcggggtcc cctatttctt ctgtttctca ctgaagtcct 1800 actgctgagc
caagactcag tcactctgga aagagcatga ccgataaaga aaacagttcc 1860
tttctgatgg ggagcgtctg agtgcagatc atgaggctct ttctctaggt ttaattcttt
1920 tccatggtga ccggacttgg tgtcttgtag cctggttacg aagtgggacg
ttgagcttct 1980 actgacgatg ccctgcatgg accagctggg atctggctgg
ggctgccctg tgtccctaac 2040 gaccataggc aatccatctt cttgtgtcag
caatttctgg acacccactg ttttccacca 2100 agagctgagg tggcaacaac
tcagtgagca ataaacaaaa tgacacagaa atgcacagtg 2160 ttgttatgaa
ggagcctgtt tacctgtgtt caaaatctgg caccattccc ttgagcaggg 2220
cccgctcagg agggaccagg tctgccagtt tctgtgcctg cagagagacg aagccccacg
2280 agccacaccc tactctacaa gaggaaaggg ggttggatgg gaagaatcta
ttttgctgtt 2340 ttggaaagca cacagccgac ctacaaacct cctgtgatgg
tgtttcttcg gatgtgtaaa 2400 ataaggcttt atttgtcaat tccgctgtaa
aataagcatt gtccgagtaa aaacagcagc 2460 aacaac 2466 86 408 DNA Homo
sapiens 86 ttttttggca tttaagtttt tcaccaattt attgctaaga ggaaacatat
aataatatgc 60 tatagggtca taaaacccac tttgcagcta tagaagcaag
ttctgcctgt gcctgtgtat 120 gtgtatgtat gacagtggac atgtaagtgt
gaaactttaa acactattac agtaagaagt 180 cttttgttga acttttgtta
gtttgagagg ctgcaatgat ttttctcctt tcaaaatgct 240 gaaatagaac
tcatcatttt gcttttcaaa ttagcaacag gtagctggtt tggaaggctg 300
gagattgatt tctctccagc tagcaagtcg tggggtcagg tcactgaagc atgtgggtga
360 tatgctgaac caccaacttg gcaaatattg aactatttta agtgcatc 408 87 431
DNA Homo sapiens misc_feature 361,431 n = A,T,C or G 87 acaaaccttc
cgggggttgc ctgagtggct gctctcggaa aagcggatcc taaataaagc 60
gggagggtta tagggcgacg tcgaggagag gacaggtctc gagtcactgc tacagtttca
120 ggtcactggg ctccgcagca gatcgtgttt tctcccgtgg ctcgagagct
gcgctggttt 180 ctcatgcaaa ctcagagccg agctaatgac atgagcaact
tttactttta cacaagatga 240 gcacgcgtgc cgaggcgctg ggcggcggct
gtgtgagttg gtggcccaga cgaacagctt 300 gtgcgagact ctgggcattt
cggtttctag atacaagatt tgcttaaatg tcacagtcca 360 nagaagtgga
tttcagtcat tgtagctact ggatgcacac aaagtaaaaa aaaaaaaact 420
tcacttgccg n 431 88 385 DNA Homo sapiens 88 gaatattcag tccacaaatt
ggcagacaat gagatttaag ccccctcctc caaactcaga 60 cattggatgg
agagtagaat ttcgacccat ggaggtgcaa ttaacagact ttgagaactc 120
tgcctatgtg gtgtttgtgg tactgctcac cagagtgatc ctttcctaca aattggattt
180 tctcattcca ctgtcaaagg ttgatgagaa catgaaggta gcacagaaaa
gagatgctgt 240 cttgcaggga atgttttatt tcaggaaaga tatttgcaaa
ggtggcaatg cagtggtgga 300 tggttgtggc aaggcccaga acagcacgga
gctcgctgca gaggagtaca ccctcatgag 360 catagacacc atcatcaatg ggaac
385 89 272 DNA Homo sapiens 89 tctttaaaat acatacgaat gtaaagagaa
aatggccaaa acctcaaaac tacgattgtt 60 gaaaacaata ttaaaaggac
acaatctaaa atcatgctac aaaaatagtg ttatcttgtt 120 taactaaatg
tacatctttt tttccaattc catgattgac aagagtgctt atgcgacgca 180
tggaaggcac cagaggtgaa gtgattattt gccttaaaat atacaaagaa ttgcctactt
240 tgaaaaagaa atagtcatac ttgtaaatga at 272 90 504 DNA Homo sapiens
90 gaagcagttt attaccttaa agcatttagc aaacctaatg tctgacctaa
tttcaaccaa 60 atgtctttat tttaccaata atcttcaaaa ctcttgattt
cccaaagcct actaaagtca 120 tgctgtcaca ggccattaga cagcatgagc
agggcaggaa agggctcttc tcccacccac 180 caggaatgtt gggtgatggc
tcagcagtta tcacattgcc tctctaaaag tcatacattg 240 gcacctaggg
tcagggagac gccatttcct gatggtccac acctattgca ctaaagtgtt 300
aattgaatgc agatgccagg gagatgcaac ttcccaggca aatgcattaa gagacaaaac
360 ggcagagtat gacctttccg tggcactcca tgggaaaagg gaagaaagcc
ttgggtgggc 420 atgtgtacaa cttcctaaac acactgcatg tgctcacctc
ccaaggatag ggagggcact 480 gtgcatgcgg gcagctcacc ctaa 504 91 467 DNA
Homo sapiens 91 tttttttttt ttttttttgc tttctcaaca aatagtttac
tcggtggaac ctaacagaac 60 taatatttct ttctgtccgt aaataaaaat
agatcatgct tgaatgtgct actttgcccg 120 aactccccaa gtcttcccgc
atcttcagtt cctccccctc caacctggtg tttatcagga 180 gaggggaaag
agcatttctt gcctggcagg aactcaagac ctagaagaaa gagggcctac 240
cctgccaagg aaacgacctt ccccttcctc gcctctgctc ctcttcccgt ttcctgtctt
300 ttccttcttt tctcctgggg tttccttctc ccgttaacta tggggacaga
cacagctatt 360 cacaagtccg tctgggcagc acactccgag gtaaggcacg
aaggtcagga gacaggttcc 420 cgtgccccaa atcctggaga agatgagtta
aagctcttcg cttcgat 467 92 229 PRT Homo sapiens 92 Met Lys Ser Ser
Asp Ile Asp Gln Asp Leu Phe Thr Asp Ser Tyr Cys 5 10 15 Lys Val Cys
Ser Ala Gln Leu Ile Ser Glu Ser Gln Arg Val Ala His 20 25 30 Tyr
Glu Ser Arg Lys His Ala Ser Lys Val Arg Leu Tyr Tyr Met Leu 35 40
45 His Pro Arg Asp Gly Gly Cys Pro Ala Lys Arg Leu Arg Ser Glu Asn
50 55 60 Gly Ser Asp Ala Asp Met Val Asp Lys Asn Lys Cys Cys Thr
Leu Cys 65 70 75 80 Asn Met Ser Phe Thr Ser Ala Val Val Ala Asp Ser
His Tyr Gln Gly 85 90 95 Lys Ile His Ala Lys Arg Leu Lys Leu Leu
Leu Gly Glu Lys Thr Pro 100 105 110 Leu Lys Thr Thr Ala Thr Pro Leu
Ser Pro Leu Lys Pro Pro Arg Met 115 120 125 Asp Thr Ala Pro Val Val
Ala Ser Pro Tyr Gln Arg Arg Asp Ser Asp 130 135 140 Arg Tyr Cys Gly
Leu Cys Ala Ala Trp Phe Asn Asn Pro Leu Met Ala 145 150 155 160 Gln
Gln His Tyr Asp Gly Lys Lys His Lys Lys Asn Ala Ala Arg Val 165 170
175 Ala Leu Leu Glu Gln Leu Gly Thr Thr Leu Asp Met Gly Glu Leu Arg
180 185 190 Gly Leu Arg Arg Asn Tyr Arg Cys Thr Ile Cys Ser Val Ser
Leu Asn 195 200 205 Ser Ile Glu Gln Tyr His Ala His Leu Lys Gly Ser
Lys His Gln Thr 210 215 220 Asn Leu Lys Asn Lys 225 93 2327 DNA
Homo sapiens 93 gggagcgaaa accaacgtgt tcggtgacag accccagcgc
cgactgagcc tctaaagcga 60 cttcagctct gccccaccaa caccaccgcg
cgcccgggaa cagccgctcc gggaagaaac 120 ctgaggggac tgcggggggc
acgagggaca gctgagggaa gggaggacgc gagagaaaca 180 gcgcaagcac
gctgagggcc gggggttgcc aggagagggg cccgcggacc cgcagagcgg 240
aggaaggtcc gggagaaaag gggcgggacg gaggagaatc cgggatcgcc tggcagaaaa
300 agagaaggga gtttctgaat cctgggaaga ggaggcgtgg gtagggatgc
ttagcccgag 360 atccgacagc agggaaccgg agcgctccgg gggaggggct
taatgctggg gaagggatgt 420 cttaaaagag gagaagcttt aaattagacg
atcggagaag gctgagggaa ttgctatgaa 480 ggggcgggag ctgaagtgta
gaggactcct ttagacagca gaaagggaaa gccgttgaga 540 agttcccttc
aaactccacc tgcctcctct ccaattcaaa ctccactccc ttctccaaaa 600
gttaaaagga aagccaagtt tgccacgctc ccctgttcct actcaataaa tacttcttct
660 actccgccac cgggaaaaca gaaaaaaaaa actaatttcc ttcccaatat
taggacttag 720 aaaagctcta ggtcccgcaa cttgaatttt agcctagggg
aatcaaaata gtaggagcat 780 tactcttgtt tcctttttca aaatcccaca
cctcatcctt cctgcgacgc catgtctacc 840 aacatttgta gtttcaagga
caggtgcgtg tccatcctgt gttgcaaatt ctgtaaacaa 900 gtgctcagct
ctaggggaat gaaggctgtt ttgctggctg atactgaaat agaccttttc 960
tctacagaca tccctcctac caacgcagtg gacttcactg gaagatgcta tttcaccaaa
1020 atctgcaaat gtaaactgaa ggacatcgca tgtttaaaat gtgggaacat
tgtaggttat 1080 catgtgattg ttccatgtag ttcctgtctt ctttcctgca
acaacggaca cttctggatg 1140 tttcacagcc aggcagttta tgatattaac
agactagact ccacaggtgt aaacgtccta 1200 ctttggggca acttgccaga
gatagaagag agtacagatg aagatgtgtt aaatatctca 1260 gcagaggagt
gtattagata aatggaatta tgatatatat gatatacaaa cttttttcta 1320
tttaaaaata tattaatgga tcaactttaa aattgttagt tgccagtgat cttttttgga
1380 aaacaaaaat ggggcatttg ttgatttatt tattttccgt ctctaattag
ttacctcagt 1440 ttgattgaag ccagtggagt tgtgcttttc ctctacttct
acttcctctc ccccaccttt 1500 ttctgcccag tgtaggtgta ttcttaaatt
cagacgggaa gattctttca catatcactc 1560 agttacctcc caatctgggg
gagtttttct tacaacttga taccagatac cattaatttt 1620 acattcctga
ataaaggcct agtacccacg catatttcaa ccatgcatat
atcaagttca 1680 actgagtttt aataggggat taaaaaaaca agctgttagg
tttccatggg cactggttct 1740 cataggttct attggtgata actgctttaa
catggagcaa gagtttgtga atcaggaaat 1800 agaataaatt aaaatttaaa
atatatagag gaatcctctt gattgctcag catgatgtta 1860 gataaatgag
tttgtcagaa aatatcagta tacgctgttt accaatgtta tttatttaca 1920
ttcttctaaa gccattatgg atattgtatt atgagagcta aacctaaata agttatcctg
1980 ttccctagga ccttctctgt aaatagtgaa ttttagacga gtagtctgtc
ctaaatctta 2040 aatagaaaaa aaaactaaag cgatttgctt aagccattgt
acattataaa gagctgtttt 2100 gttttgcttt gctttgcttt gttttgtttt
ttttaaagct gcattcagag ccacaaagga 2160 ataggaaagt agggtagtgt
tggattctgg ttttatgtaa ctctaaaata aatgtatctc 2220 tttaatatct
cagttgtagg gattttgtca ataccaaagc agactgagtt gtggttttgt 2280
aaataaagtt ttttctaaaa atgaaaaaaa aagaaaaaaa aaaaaaa 2327 94 2370
DNA Homo sapiens misc_feature 741,1195,1683,2360 n = A,T,C or G 94
gggagcgaaa accaacgtgt tcggtgacag accccagcgc cgactgagcc tctaaagcga
60 cttcagctct gccccaccaa caccaccgcg cgcccgggaa cagccgctcc
gggaagaaac 120 ctgaggggac tgcggggggc acgagggaca gctgagggaa
gggaggacgc gagagaaaca 180 gcgcaagcac gctgagggcc gggggttgcc
aggagagggg cccgcggacc cgcagagcgg 240 aggaaggtcc gggagaaaag
gggcgggacg gaggagaatc cgggatcgcc tggcagaaaa 300 agagaaggga
gtttctgaat cctgggaaga ggaggcgtgg gtagggatgc ttagcccgag 360
atccgacagc agggaaccgg agcgctccgg gggaggggct taatgctggg gaagggatgt
420 cttaaaagag gagaagcttt aaattagacg atcggagaag gctgagggaa
ttgctatgaa 480 ggggcgggag ctgaagtgta gaggactcct ttagacagca
gaaagggaaa gccgttgaga 540 agttcccttc aaactccacc tgcctcctct
ccaattcaaa ctccactccc ttctccaaaa 600 gttaaaagga aagccaagtt
tgccacgctc ccctgttcct actcaataaa tacttcttct 660 actccgccac
cgggaaaaca gaaaaaaaaa actaatttcc ttcccaatat taggacttag 720
aaaagctcta ggtcccgcaa yttgaatttt agcctagggg aatcaaaata gtaggagcat
780 tactcttgtt tcctttttca aaatcccaca cctcatcctt cctgcgacgc
catgtctacc 840 aacatttgta gtttcaagga caggtgcgtg tccatcctgt
gttgcaaatt ctgtaaacaa 900 gtgctcagct ctaggggaat gaaggctgtt
ttgctggctg atactgaaat agaccttttc 960 tctacagaca tccctcctac
caacgcagtg gacttcactg gaagatgcta tttcaccaaa 1020 atctgcaaat
gtaaactgaa ggacatcgca tgtttaaaat gtgggaacat tgtaggttat 1080
catgtgattg ttccatgtag ttcctgtctt ctttcctgca acaacggaca cttctggatg
1140 tttcacagcc aggcagttta tgatattaac agactagact ccacaggtgt
aaacrtccta 1200 ctttggggca acttgccaga gatagaagag agtacagatg
aagatgtgtt aaatatctca 1260 gcagaggagt gtattagata aatggaatta
tgatatatat gatatacaaa cttttttcta 1320 tttaaaaata tattaatgga
tcaactttaa aattgttagt tgccagtgat cttttttgga 1380 aaacaaaaat
ggggcatttg ttgatttatt tattttccgt ctctaattag ttacctcagt 1440
ttgattgaag ccagtggagt tgtgcttttc ctctacttct acttcctctc ccccaccttt
1500 ttctgcccag tgtaggtgta ttcttaaatt cagacgggaa gattctttca
catatcactc 1560 agttacctcc caatctgggg gagtttttct tacaacttga
taccagatac cattaatttt 1620 acattcctga ataaaggcct agtacccacg
catatttcaa ccatgcatat atcaagttca 1680 acygagtttt aataggggat
taaaaaaaca agctgttagg tttccatggg cactggttct 1740 cataggttct
attggtgata actgctttaa catggagcaa gagtttgtga atcaggaaat 1800
agaataaatt aaaatttaaa atatatagag gaatcctctt gattgctcag catgatgtta
1860 gataaatgag tttgtcagaa aatatcagta tacgctgttt accaatgtta
tttatttaca 1920 ttcttctaaa gccattatgg atattgtatt atgagagcta
aacctaaata agttatcctg 1980 ttccctagga ccttctctgt aaatagtgaa
ttttagacga gtagtctgtc ctaaatctta 2040 aatagaaaaa aaaactaaag
cgatttgctt aagccattgt acattataaa gagctgtttt 2100 gttttgcttt
gctttgcttt gttttgtttt ttttaaagct gcattcagag ccacaaagga 2160
ataggaaagt agggtagtgt tggattctgg ttttatgtaa ctctacccta ctttcctatt
2220 cctttgtgtc ctgtaacttt ttttacctat caatatgagt tgctgtgctt
cagtgtgtat 2280 tttttaagtt gctgggcatt acacttacca attaaagaat
tttggaaatt caaaaaaaaa 2340 aaaaaaaaaa aaaaaaaaam aaaaaaaaaa 2370 95
450 DNA Homo sapiens 95 atgtctacca acatttgtag tttcaaggac aggtgcgtgt
ccatcctgtg ttgcaaattc 60 tgtaaacaag tgctcagctc taggggaatg
aaggctgttt tgctggctga tactgaaata 120 gaccttttct ctacagacat
ccctcctacc aacgcagtgg acttcactgg aagatgctat 180 ttcaccaaaa
tctgcaaatg taaactgaag gacatcgcat gtttaaaatg tgggaacatt 240
gtaggttatc atgtgattgt tccatgtagt tcctgtcttc tttcctgcaa caacggacac
300 ttctggatgt ttcacagcca ggcagtttat gatattaaca gactagactc
cacaggtgta 360 aacgtcctac tttggggcaa cttgccagag atagaagaga
gtacagatga agatgtgtta 420 aatatctcag cagaggagtg tattagataa 450 96
149 PRT Homo sapiens 96 Met Ser Thr Asn Ile Cys Ser Phe Lys Asp Arg
Cys Val Ser Ile Leu 5 10 15 Cys Cys Lys Phe Cys Lys Gln Val Leu Ser
Ser Arg Gly Met Lys Ala 20 25 30 Val Leu Leu Ala Asp Thr Glu Ile
Asp Leu Phe Ser Thr Asp Ile Pro 35 40 45 Pro Thr Asn Ala Val Asp
Phe Thr Gly Arg Cys Tyr Phe Thr Lys Ile 50 55 60 Cys Lys Cys Lys
Leu Lys Asp Ile Ala Cys Leu Lys Cys Gly Asn Ile 65 70 75 80 Val Gly
Tyr His Val Ile Val Pro Cys Ser Ser Cys Leu Leu Ser Cys 85 90 95
Asn Asn Gly His Phe Trp Met Phe His Ser Gln Ala Val Tyr Asp Ile 100
105 110 Asn Arg Leu Asp Ser Thr Gly Val Asn Val Leu Leu Trp Gly Asn
Leu 115 120 125 Pro Glu Ile Glu Glu Ser Thr Asp Glu Asp Val Leu Asn
Ile Ser Ala 130 135 140 Glu Glu Cys Ile Arg 145
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